Production of eosinophil-activating factor (EAF) by peripheral blood mononuclear cells from asthma patients

Mononuclear cells from blood samples from asthmatic patients were tested for their ability to produce two eosinophil activating factors, EAF and TNF. High levels of EAF were produced by some but not by all patients. The influence of external factors on EAF production was evaluated. Patients receiving prednisolone (10-30 mg/day) tended to produce only low levels of EAF. Prednisolone has been shown to inhibit production of EAF in vitro by isolated mononuclear cells, at concentrations of 0.1-1 micrograms steroid/ml. Incubation of mononuclear cells with certain allergens enhances EAF production in sensitive individuals. Little or no production of TNF was observed in the patients examined.     

Detection and neutralization of bovine tumor necrosis factor by a monoclonal antibody.

Monoclonal antibodies (MAbs) have been produced which are specific for bovine tumor necrosis factor (TNF). MAb BC9 detects bovine TNF in a radioimmunoassay with a detection limit of 24 pg/ml. BC9 also neutralizes the in vitro biological function of bovine recombinant TNF. The activity of 250 ng TNF/ml was entirely neutralized by 1% ascitic fluid. When ascites was added at a saturating concentration (10% ascitic fluid), up to 25 micrograms TNF per ml was neutralized. The neutralizing effect of BC9 was seen in cytotoxic assays using L929 cells and WEHI 164 clone 13 cells. The cytotoxic activity of supernatants from in vitro activated bovine monocytes was entirely blocked by BC9.     

A new method for measuring eosinophil activating factors, based on the increased expression of CR3 alpha chain (CD11b) on the surface of activated eosinophils

The observation that activation of eosinophils in vitro with PAF increases the surface expression of the alpha chain of the complement receptor CR3 (CD11b) has been extended to other eosinophil activating factors. CD11b may be detected on activated eosinophils by reaction with mouse monoclonal anti-human CD11b IgG, following the addition of urease-conjugated sheep anti-mouse IgG. CD11b levels were increased on eosinophils after incubation with (a) recombinant colony stimulating factors, IL-3, GM-CSF and IL-5, at concentrations of 100 U/ml, or (b) with eosinophil activating factors, recombinant TNF alpha (1000 U/ml), EAF purified from mononuclear cell supernatants and PAF (10(-6) M). CD11b levels were not affected by IL-1 alpha, IL-2 or IFN-gamma. Unstimulated neutrophils had higher levels of CD11b than unstimulated eosinophils, but neutrophil CD11b was unaffected by IL-3, GM-CSF and IL-5 and was only slightly affected by TNF, EAF and PAF. Polyclonal rabbit antibodies to IL-3 and TNF neutralised their CD11b enhancing activities. The PAF antagonists WEB 2086 and WEB 2170 neutralised the CD11b enhancing activity of PAF. We conclude that measurement of CD11b expression on eosinophils is a convenient method for the assay of eosinophil activating activity.     

Factors secreted by untreated and hydrocortisone-treated monocytes that modulate neutrophil function

When incubated for 1.0 hr with neutrophils isolated from bovine peripheral blood, hydrocortisone (0.05, 0.5, or 5.0 micrograms/ml) had no significant effect on their ability to ingest Staphylococcus aureus, to reduce nitroblue tetrazolium (NBT) or iodinate protein, or to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against chicken erythrocytes. Random migration was significantly enhanced by the highest concentration of hydrocortisone (HC) but was unaffected by the lower concentrations. Resting bovine monocytes were incubated for 24 hr in medium with or without added hydrocortisone (0.05, 0.5, or 5.0 micrograms/ml). Purified bovine neutrophils were then incubated for 1.0 hr with the resulting monocyte supernatant (MS). The MS from untreated monocytes significantly enhanced neutrophil ADCC but did not affect the other neutrophil functions evaluated. Supernatants from monocytes incubated with 0.5 or 5.0 micrograms/ml hydrocortisone (HC-treated MS) failed to enhance neutrophil ADCC but did enhance neutrophil random migration under agarose. The other parameters of neutrophil function were unaffected. Production of the factors in MS was reduced by inhibition of protein synthesis in monocytes. Their activity was reduced by exposure of MS to proteolytic enzymes, suggesting that the monocyte factors were polypeptides or proteins. Protein synthesis by the neutrophil was not necessary for its response to the monocyte-produced ADCC-enhancing factor but was necessary for its response to the HC-induced monocyte factor. The results suggest that the antiinflammatory effects of glucocorticoids may be partially mediated by factors released by monocytes.     

Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo.

Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin.     

Partial purification and biological properties of an eosinophil-activating factor

A protein (eosinophil-activating factor, EAF), which enhances the capacity of human peripheral blood eosinophils to kill antibody-coated schistosomula of Schistosoma mansoni, has been partially purified from supernatants of cultured peripheral blood mononuclear cells by sequential chromatography on Sephacryl S200 and DEAE-cellulose. This protein is acidic with a molecular mass on gel filtration of 40 +/- 7 kDa. It not only enhances the activity of eosinophils against schistosomula but also increases their ability to lyse antibody-coated, herpes simplex virus-infected Chang liver cells. It enhances the production of superoxide and hydrogen peroxide by eosinophils that occurs both spontaneously and in response to opsonized zymosan. However, increased respiratory burst activity does not appear to be responsible for the enhancement of eosinophil-mediated killing of schistosomula, since a comparable or greater increase in hydrogen peroxide production is induced by column fractions that have little or no effect on schistosomulum killing. EAF enhances eosinophil degranulation, both spontaneously and after incubation with opsonized zymosan. Enhanced degranulation is associated with release of eosinophil peroxidase and eosinophil cationic protein. These findings suggest that EAF enhances the capacity of eosinophils to kill parasites by increasing the extent of eosinophil degranulation and the amount of toxic granule proteins that are secreted.     

The effects of eosinophil activating factor on IgG-dependent sulphidopeptide leukotriene generation by human eosinophils.

Eosinophil activating factor (EAF) is a 40 kD protein released from cultured, unstimulated human monocytes which enhances the IgG-dependent eosinophil-mediated cytotoxicity of helminthic larvae. We have recently shown that eosinophils elaborate substantial quantities of leukotriene C4 (LTC4) during incubation with IgG-coated particles and now report that EAF, partially purified by sequential chromatography on Sephacryl S-200 and DEAE-cellulose, enhanced this IgG-dependent LTC4 production by human eosinophils in a dose- and time-dependent fashion. LTC4 production by normal density eosinophils, separated on discontinuous metrizamide gradients, was significantly increased after incubation with several dilutions of EAF (P less than 0.05), although an increase was not seen with low density cells. The enhancement was similar in degree to that seen when normal density eosinophils were activated with the bacterial analogue, f-met-leu-phe (fMLP). EAF produced a time-dependent increase in LTC4 which was significantly greater (P less than 0.01) than the control. Sulphidopeptide leukotriene (LT) generation was validated by reverse phase high pressure liquid chromatography (RP-HPLC). These results indicate that there is a firm association between monocytes, eosinophils and LTC4; an observation which may be of relevance to mechanisms in chronic asthma and related disorders, and in immune reactions against migrating helminthic larvae.     

Changes in the pharmacokinetics of theophylline during estrus in rats

The influence of menstrual cycle on drug kinetics is not largely documented. Concerning theophylline it was of interest to investigate whether or not the kinetics of this drug is modified throughout the oestrous cycle. Though the kinetics of this drug was assessed in adult Wistar female rats at different stages of the oestrous cycle: proestrous (P), oestrous (O) and diestrus (D). Our preliminary data clearly indicate statistically significant differences (p less than 0.01) of theophylline kinetic parameters such as elimination half-life: 8.70 +/- 0.60 h (P), 4.61 +/- 0.16 h (O) and 5.01 +/- 0.85 h (D), area under concentration versus time curve (AUC): 214.61 +/- 3.58 micrograms.h/ml (P), 128.64 +/- 9.64 micrograms.h/ml (O) and 165.57 +/- 23.86 micrograms.h/ml (D). These results agree with clinical data reported on the influence of estroprogestative drugs on theophylline kinetics in women (higher elimination half-life and lower clearance). In order to explain some of our findings the possible variations of theophylline protein binding and metabolism throughout the oestrous cycle are under investigation.     

In vitro suppression of fibroblast growth inhibitory lymphokine production by asbestos.

Human peripheral blood mononuclear leucocytes (PBML) stimulated with concanavalin A (Con A) or phytohaemagglutinin (PHA) produced a soluble factor which inhibits lung fibroblast DNA synthesis and growth. Lymphocyte enriched preparations produced significant growth inhibitory activity in the presence of PHA whereas media from adherent mononuclear cells incubated in the presence of the mitogen did not contain similar activity. This fibroblast growth inhibitory factor (FGIF) was non-dialysable, heat stable and resistant to pH 5. FGIF was also resistant to treatment with chymotrypsin and phosphodiesterase but partially sensitive to treatment with trypsin. Interestingly, there was significant suppression of FGIF production by PBML cultured with PHA in the presence of low concentrations of chrysotile asbestos (5-25 micrograms/ml). In this regard, asbestos (25 micrograms/ml) was not cytotoxic for lymphocytes but had a damaging effect on monocytes as evidenced by the release of lactate dehydrogenase (LDH) a cytoplasmic enzyme, in their culture media. These findings indicate that stimulated lymphocytes have the ability to inhibit fibroblast proliferation by releasing FGIF and that asbestos interfere with this process. Thus, while FGIF may regulate the extent of connective tissue proliferation during normal repair process, suppression of its production by asbestos may contribute to excessive fibroblast accumulation and fibrosis.     

Theophylline administration in children with asthma: optimal pulmonary function and possible tolerance to chronic administration

This study was performed to assess the need of obtaining serum theophylline (T) levels between 10--20 micrograms/ml to achieve maximum reversibility of airway obstruction in chronic childhood asthma. Twenty-seven children with daily asthma (ages 9--16 years mean 11.7) were studied to determine the serum T levels required to obtain optimal pulmonary function tests as measured by FEV1 and FEF25--75. Two parallel groups were created. Group 1 (13 subjects) received rapid release (RR) anhydrous T. Group 2 (14 subjects) received sustained release (SR) anhydrous T. The groups were identical in age, weight, height and PFT: p = values greater than or equal to .21 (t test for equivalent means). During initial titration maximal PFT's were obtained in Group 1 subjects with mean T level of 7.1 micrograms/ml and in Group 2 subjects with mean T level of 8.5 micrograms/ml. The PFT responses and theophylline dose responses of each group were not significantly different from each other. After two months of continuous high dose (10--20 micrograms/ml) therapy each subject was again titrated for dose response of PFT with serum T levels. PFT's were not significantly different from the acute studies. After continuous high dose theophylline therapy serum T levels needed (mean 11.5 micrograms/ml) to obtain the maximal PFT response were significantly higher than during the initial titration (mean 7.1 to 8.5 micrograms/ml). Maximal PFT's in many asthmatic children do not require serum T greater than or equal to 10 micrograms/ml in an acute dosage. The continuous use of high dose theophylline may lead to tolerance, thus requiring a higher theophylline dose and subsequent serum level to obtain maximal PFT.     

Pharmacokinetics and pharmacodynamics of sustained-release theophylline formulation, Slo-bid, in both single and multiple dosing studies of asthmatic children

The pharmacokinetics of theophylline in serum and saliva, and the pulmonary functions were examined in seven asthmatic children aged 9-14 years in both single (6.7 +/- 0.8 micrograms/kg) and multiple dosing settings of sustained-release theophylline formulation, Slo-bid, designed for 12-hour dosing intervals. In the single dose study, the mean maximum concentration (Cmax) and the time taken to reach maximum concentration (Tmax) in serum were 5.5 micrograms/ml and 7.3 hours, and the same parameters in saliva were 3.8 micrograms/ml and 7.3 hours. In the multiple dose study which lasted five days, Cmax, the mean minimum concentration (Cmin), the peak-trough fluctuation (delta P-T) and Tmax in plasma were 13.2 micrograms/ml, 9.9 micrograms/ml, 3.3 micrograms/ml and 5.4 hours, and those in saliva were 10.4 micrograms/ml, 7.0 micrograms/ml, 3.4 micrograms/ml and 5.0 hours, respectively. These results, showing a significant correlation, in both single and multiple dose trials, between the theophylline concentration in serum and the improvement of pulmonary function are support the contention that the therapeutic range of theophylline concentration in serum is 5 to 20 micrograms/ml. There was a significant correlation between the concentration in serum and saliva (r = 0.943, p less than 0.001). The predicted concentrations in serum from those in saliva were +/- 3 micrograms/ml of the measured concentrations in 92.8% of measurements. From these results, we concluded that the sustained-release formulation of theophylline, Slo-bid, is suitable for the treatment in asthmatic children.     

Rat eosinophil-mediated antibody-dependent cellular cytotoxicity: investigations of the mechanisms of target cell lysis and inhibition by glucocorticoids.

Purified eosinophils from the peritoneal washings of N. brasiliensis infected rats demonstrated antibody-dependent cellular cytotoxicity (ADCC) for 51Cr-labelled chicken erythrocytes. The F(ab')2 fragment of the antibody did not support cytotoxicity thereby demonstrating the importance of the eosinophil Fc receptor to this activity. Bystander lysis of erythrocytes did not occur, indicating that the eosinophil does not release lytic agents free into the medium. Cytochalasin B (1.25-5 micrograms/ml) colchicine (10(-5)-10(-3) M) and chloroquine (10(-4)-10(-3) M) inhibited eosinophil ADCC. Inhibition was also demonstrated by methylprednisolone, 10(-7)-10(-3) M and this inhibition was blocked by the protein synthesis inhibitor, cycloheximide (25 micrograms/ml). Cycloheximide alone had no effect. This block of steroid inhibition by cycloheximide suggests that the steroid effect on this system may be mediated by a newly synthesized protein and implies that the eosinophil may possess a glucocorticoid receptor.     

Decreased monocyte-mediated angiogenesis in scleroderma.

Scleroderma is a disease characterized by proliferative vascular lesions in which monocytes/macrophages may play a key role. Monocytes were isolated from 14 scleroderma patients and 11 normal controls and cultured with or without lipopolysaccharide (LPS) (5 micrograms/ml). Monocyte-conditioned medium was assayed in the rat corneal bioassay for angiogenesis. Conditioned medium from normal monocytes was nonangiogenic, as was conditioned medium from scleroderma monocytes. While conditioned medium from LPS-activated normal monocytes was potently angiogenic in 11/13 corneas, conditioned medium from LPS-activated scleroderma monocytes was angiogenic in only 3/14 corneas. Levels of the angiogenic cytokine tumor necrosis factor-alpha (TNF-alpha) were measured in conditioned medium from scleroderma and normal monocytes. TNF-alpha levels were not significantly different in patient and control groups and thus do not account for the decreased angiogenic activity exhibited by scleroderma monocytes. As monocytes require activation to produce angiogenic activity, we determined the cell surface binding of monoclonal antibodies to activation-related (HLA-DR, 3D8, and 8D7) and other (Leu-M5) markers on monocytes by radioimmunoassay. Monocytes were cultured alone, with LPS (5 micrograms/ml), or with interferon-gamma (IFN) (200 units/ml). The usual increase in binding of anti-HLA-DR on stimulation of scleroderma monocytes with IFN was slightly less than that of controls. IFN-stimulated monocytes bound less anti-8D7 than controls. Anti-3D8 and anti-Leu-M5 binding was comparable in both groups. These results suggest that scleroderma monocytes do not produce normal levels of angiogenic activity with LPS stimulation, have some altered markers of activation on their cell surfaces, and may thus contribute to the aberrant vascular proliferation found in this disease.     

Non-immunological recognition and killing of xenogeneic cells by macrophages. II. Mechanism of killing.

Macrophages are cytotoxic to chicken embryonic fibroblasts without either previous activation or lymphocyte assistance. This cytotoxic activity (xenolysis) is expressed by non-activated macrophages from athymic mice as well as by pure macrophage populations. Neither macrophage lysate nor supernatants of macrophages cultivated with fibroblasts cause xenolysis. Unlike macrophage tumoricidal activity, killing of xenogeneic cells is not dependent on specific serum factors and is expressed by macrophages from a lipopolysaccharide (LPS) unresponsive strain (C3H/HeJ). Xenolysis is expressed also by trypsin-treated macrophages and by macrophages from 5-day-old cultures. Killing of chicken fibroblasts by macrophages is not affected by hydrocortisone (100 micrograms/ml) gold salt (1 mg/ml) and colchicine (100 micrograms/ml). On the other hand, cytochalasin B (10 micrograms/ml) completely abolishes the killing, probably by interfering with macrophage mobility and extension of filopodia toward the targets. It is suggested that the xenolytic activity of macrophages represents a primitive trait of phagocytes which assists the body in defence against multicellular parasites.     

Long-term prognosis of asthmatic patients treated with low-dose beclomethasone dipropionate]

To clarify the prognosis of asthmatics treated with low-dose of inhaled beclomethasone dipropionate (BDP), we retrospectively assessed 43 patients treated with initial dose of 200 or 400 micrograms/day for 5 years, and obtained the following results. 1) 15 patients achieved step-down therapy (group A), 17 patients maintained initial dose of BDP (group B), and 11 patients required step-up therapy of BDP or daily use of oral prednisolone (group C). 2) There was no significant difference in age, sex, duration of disease, severity of disease, peripheral eosinophil counts, %FEV1 and histamine PC20 before BDP treatment among three groups. The percentage of atopic asthmatics was significantly higher in group C than in group A. 3) There was no significant difference in symptom and histamine PC20 between after 1 year state and after 5 years state in three groups. 4) After 1 year from the start of BDP treatment, only 18% patients got symptom free and neither patients exceeded 20,000 micrograms/ml of histamine PC20 in group C. Long-term treatment of low-dose BDP inhalation was effective on mild/moderate asthmatics. Patients requiring step-up therapy had not got sufficient improvement in bronchial hyperresponsiveness after one-year treatment.     

Release of cytokines induced by Salmonella typhimurium porins.

Salmonella typhimurium SH5014 porins induce the release of tumor necrosis factor alpha (TNF-alpha), interleukin 1 alpha (IL-1 alpha), and IL-6 by human monocytes and of gamma interferon (IFN-gamma) and IL-4 by human lymphocytes. Porins at 1 microgram/ml induce the greatest release of TNF-alpha, IL-1 alpha, and IL-6 by monocytes and of IL-4 by lymphocytes, while porins at 5 micrograms/ml induce the greatest release of IFN-gamma by lymphocytes. The R form of lipopolysaccharide (LPS-R) induces the greatest release of TNF-alpha and IL-1 alpha by monocytes when used at a low concentration (1 microgram/ml). At higher concentrations (5 and 10 micrograms/ml, respectively), LPS-R induces the maximal release of IL-6 from monocytes and the maximal release of IL-4 from lymphocytes. The S form of LPS (LPS-S) induces the greatest release of TNF-alpha, IL-1 alpha, and IL-6 by monocytes and that of IL-4 by lymphocytes when used at a concentration of 1 microgram/ml. After stimulation with LPS-S, the largest quantity of TNF-alpha and IL-1 alpha released was less than that obtained after stimulation with LPS-R at the same concentration, while the quantity of IL-6 released was found to be slightly higher than that obtained after stimulation with porins or LPS-R. LPS-S (1 microgram/ml) induces IFN-gamma release from lymphocytes in notably smaller quantities than that obtained with LPS-R and slightly larger quantities than that obtained with porins. The preparation of porins used was found to be contaminated with 10 pg of LPS per 10 micrograms of porins, a quantity which was found to have no biological effect; furthermore, porin preparations with the addition of polymyxin B gave the same results.     

Induction of tumor necrosis factor and interleukin-1 by purified staphylococcal toxic shock syndrome toxin 1 requires the presence of both monocytes and T lymphocytes.

Highly purified staphylococcal toxic shock syndrome toxin 1 (TSST-1) was tested for its ability to induce the cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) from fractionated human peripheral blood mononuclear cells prepared from seven healthy donors. Highly purified monocytes alone or T lymphocytes alone did not produce TNF or IL-1 when incubated with TSST-1 at 37 degrees C for up to 72 h. However, the addition of 10 micrograms of TSST-1 per ml to a 1:1 mixture of monocytes and T cells resulted in significant TNF (predominantly TNF-alpha) and IL-1 beta production after 24 h at 37 degrees C. The nature of the monocyte/T-cell interaction did not appear to involve gamma interferon (IFN-gamma), since 10 micrograms of rabbit anti-IFN-gamma per ml did not neutralize TNF-alpha production after TSST-1 induction. Similarly, L243, a monoclonal antibody to HLA-DR which blocks TSST-1 binding to monocytes, did not inhibit TNF-alpha production following TSST-1 induction. However, direct contact between monocytes and T cells was required, since physical separation of cells in double-chamber culture wells abolished TNF-alpha secretion after TSST-1 stimulation. Furthermore, paraformaldehyde fixation of either monocytes or T cells prior to addition to viable T cells or monocytes, respectively, also abolished TNF-alpha secretion, suggesting that aside from cell contact, soluble factors were also involved. Our results suggest that cytokine production involves more than binding of TSST-1 to its receptor on monocytes alone and that cell contact with T cells and the release of a soluble factor(s) other than IFN-gamma may be essential for the induction of cytokines by this toxin.     

Effect of a 4-week treatment with theophylline on sputum eosinophilia and sputum eosinophil chemotactic activity in steroid-naive asthmatics

BACKGROUND: The precise mechanism of action of theophylline in asthma is not fully understood but recent data have drawn attention to its potential anti-inflammatory effect. OBJECTIVE: The purpose of this study was to assess the effect of theophylline on sputum eosinophilia and sputum eosinophil chemotactic activity in steroid-naive asthmatics. METHOD: We performed a 4-week randomized double-blind, placebo-controlled, parallel group study in 21 mild to moderate steroid-naive asthmatics whose sputum eosinophilia was found twice > 5% during the run in period. Eleven subjects received 600 mg/24 h theophylline for the first 2 weeks and 900 mg/24 h for the last 2 weeks while 10 subjects took a placebo for 4 weeks. Sputum was induced after 2 and 4 weeks of treatment and 1 week after stopping the treatment. The sputum samples were compared for their cell counts, eosinophil cationic protein (ECP) levels and eosinophil chemotactic activity using micro-Boyden chambers. RESULTS: Serum theophylline concentrations reached 7 and 11 microg/mL at V3 and V4, respectively. Intragroup comparisons showed that theophylline, but not placebo, caused a significant reduction in sputum eosinophil counts at V3 (62 +/- 10% from baseline, P < 0.01) and a strong trend at V4 (67 +/- 16% from baseline, P = 0.07) when compared to baseline. The intergroup difference obtained after comparing the area under the curve over the 4 week treatment period only approached the statistical significance (P = 0.08). At baseline the fluid phase of the sputum contained a significant eosinophil chemotactic activity which was inhibited after a 4-week treatment by theophylline (P < 0. 01) but not by placebo. The mean sputum theophylline levels after 4 weeks of treament (1.7 microg/mL) was lower than that required to cause significant inhibition of eosinophil chemotaxis in vitro. CONCLUSION: Theophylline decreases the natural sputum eosinophil chemotactic activity present in asthmatics. However, when using a small sample size, the 35% reduction in sputum eosinophilia achieved by theophylline failed to reach statistical significance when compared to that seen after placebo.     

Comparative activity of doxorubicin and its major metabolite, doxorubicinol, on V79/AP4 fibroblasts: a morphofunctional study.

Doxorubicin (DXR), an anthracycline antineoplastic drug, is mainly metabolized to the C-13 dihydroderivative doxorubicinol (DXR-ol), which displays cytotoxic activity on various cell lines. To better characterize the cytotoxic activity of this metabolite, we have studied the effect of DXR (0.1-10 micrograms/ml) or DXR-ol (1-100 micrograms/ml) on the transformed fibroblast cell line V79/AP4 by means of the clonogenic assay, cytofluorescence, and light and electron microscopy. Both DXR and DXR-ol displayed a dose-dependent inhibition of colony formation with an IC50 factor DXR-ol/DXR of 19.5. A striking nuclear fluorescence was observed after DXR but not after DXR-ol. A low number of mitoses and a decrease in nucleoli staining affinity were the most evident alterations induced by DXR. Electron microscopy showed both nuclear and cytoplasmic changes in DXR treated cells: nucleolar segregation, cytoplasmic vacuoles, and mitochondrial swelling with dense needle-shaped material were observed. Exposure to formic acid confirmed the calcific nature of the mitochondrial bodies. Only the highest dose of DXR-ol brought about nuclear and cytoplasmic ultrastructural changes similar to those induced by DXR. Our data describe new in vitro findings on the cytotoxicity and morphological alterations induced by both DXR and DXR-ol, with a lower activity of DXR-ol against V79/AP4 fibroblasts.     

Induction of release of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans.

The role of cytokines in gram-positive infections is still relatively poorly defined. The purpose of this study was to establish whether or not intact staphylococci and purified peptidoglycans and peptidoglycan components derived from staphylococci are capable of stimulating the release of tumor necrosis factor (TNF) by human monocytes. We show here that intact staphylococci and purified peptidoglycans, isolated from three Staphylococcus epidermidis and three S. aureus strains, were indeed able to induce secretion of TNF by human monocytes in a concentration-dependent fashion. TNF release was detected by both enzyme immunoassay and the L929 fibroblast bioassay. In the enzyme immunoassay, a minimal concentration of peptidoglycan of 1 micrograms/ml was required to detect TNF release by monocytes, whereas in the bioassay a peptidoglycan concentration of 10 micrograms/ml was needed to detect a similar amount of TNF release. Peptidoglycan components such as the stem peptide, tetra- and pentaglycine, and muramyl dipeptide were unable to induce TNF release from human monocytes.