Increased leukocyte aggregation induced by gamma-globulin: a clue to the presence of pseudoleukopenia.

BACKGROUND: Previous articles have reported the appearance of leukopenia after the administration of high-dose intravenous gamma-globulins. METHODS: A simple slide test was used to reveal the state of leukocyte aggregation (LA) in the peripheral blood as well as the absolute number of leukocytes in the slides. RESULTS: A significant (P < 0.00001) increment was noted in the state of LA, from 9 +/- 6% before to 25 +/- 11% after gamma-globulin administration. This was accompanied by a significant (P < 0.0004) reduction in the number of white blood cells in the peripheral blood (from 8433 +/- 3905 to 6550 +/- 3252 cells/mm3) but no significant change in the absolute number of the cells as determined by the leukocyte count per high power field in the peripheral slides. CONCLUSIONS: We raise the possibility that the leukopenia reported in some patients after the intravenous administration of high dose gamma-globulin is explained in part by the agglutination effect of the drug.

Hematological toxicity of doxorubicin-containing protocols in dogs with spontaneously occurring malignant tumors.

The medical records of 49 dogs with spontaneously occurring malignant tumors treated with doxorubicin-based chemotherapy protocols were evaluated for hematological toxicity. Protocols included vincristine, doxorubicin, and cyclophosphamide (VAC); 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC); doxorubicin and cyclophosphamide (AC); and doxorubicin and dacarbazine (ADIC). Prevalence of Grades 1, 2, or 3 toxicities were less than 30%, and the prevalence of Grade 4 toxicity alone was less than 5%. The frequency of sepsis was less than 2.5% in dogs treated with VAC, FAC, or AC, and it was 15% in dogs treated with ADIC. There were no significant differences in the prevalence or severity of hematological toxicity caused by VAC or AC. Five-fluorouracil, doxorubicin, and cyclophosphamide caused significantly more severe neutropenia than VAC or AC. The low prevalence of hematological complications makes these protocols acceptable for use in practice.

The pharmacokinetics and pharmacodynamics of GW395058, a peptide agonist of the thrombopoietin receptor, in the dog, a large-animal model of chemotherapy-induced thrombocytopenia.

GW395058, a PEGylated peptide agonist of the thrombopoietin receptor, stimulates megakaryocytopoiesis and has previously been shown to increase platelet counts in vivo. The pharmacokinetics and pharmacodynamics of GW395058 were characterized using a randomized, crossover study in a large-animal model (dog) of chemotherapy-induced thrombocytopenia. Nine beagle dogs received i.v. carboplatin (350 mg/m(2)) on day 0 and day 28. GW395058 (1.31 mg/kg) (n = 6) or vehicle control (n = 3) was administered on day 1 and day 29 either as an i.v. bolus or s.c. injection. After i.v. administration, peak concentrations of GW395058 occurred rapidly, while the half-life averaged approximately 56 h. Bioavailability (+/- standard deviation) of GW395058 given s.c. was 78.2% (20.9%). GW395058 (i.v. and s.c.) ameliorated the platelet nadir (p = 0.0086) and resulted in a shorter time to recovery compared to the control group. The mean nadir platelet counts following carboplatin administration were 197,000 cells/microl (80,000) for the i.v. GW395058-dose group, 183,000 cells/microl (72,000) for the s.c.-dose group and 71,000 cells/microl (38,000) for the vehicle-alone group. GW395058 reduced the thrombocytopenic effects of carboplatin in dogs. No GW395058-related adverse side effects were observed.

Pharmacokinetic and pharmacodynamic study of the combination of docetaxel and topotecan in patients with solid tumors.

PURPOSE: The sequence in which chemotherapeutic agents are administered can alter their pharmacokinetics, therapeutic effect, and toxicity. We evaluated the pharmacokinetics and pharmacodynamics of docetaxel and topotecan when coadministered on two different sequences of administration. PATIENTS AND METHODS: On cycle 1, docetaxel was administered as a 1-hour infusion at 60 mg/m(2) without filgrastim and at 60, 70, and 80 mg/m(2) with filgrastim on day 1, and topotecan was administered at 0.75 mg/m(2) as a 0.5-hour infusion on days 1 to 4. On cycle 2, topotecan was administered on days 1 to 4, and docetaxel was administered on day 4. Cycles were repeated every 21 days. Blood samples for high-performance liquid chromatography measurement of docetaxel (CL(DOC)) and topotecan (CL(TPT)) total clearance were obtained on day 1 of cycle 1 and day 4 of cycle 2. CL(DOC) and CL(TPT) were calculated using compartmental methods. RESULTS: Mean +/- SD CL(DOC) in cycles 1 and 2 were 75.9 +/- 79.6 L/h/m(2) and 29.2 +/- 17.3 L/h/m(2), respectively (P: <.046). Mean +/- SD CL(TPT) in cycles 1 and 2 were 8.5 +/- 4.4 L/h/m(2) and 9.3 +/- 3.4 L/h/m(2), respectively (P: >. 05). Mean +/- SD neutrophil nadir in cycles 1 and 2 were 4,857 +/- 6, 738/microL and 2,808 +/- 4,518/microL, respectively (P: =.02). CONCLUSION: Administration of topotecan on days 1 to 4 and docetaxel on day 4 resulted in an approximately 50% decrease in docetaxel clearance and was associated with increased neutropenia.

The effect of long-term treatment with granulocyte colony-stimulating factor on hematopoiesis in HIV-infected individuals.

This randomized, placebo-controlled trial examine the long-term effect of granulocyte colony-stimulating factor (G-CSF) on absolute numbers of CD34+ progenitor cells and progenitor cell function in human immunodeficiency virus (HIV)-infected patients. G-CSF (300 microg filgrastim) or placebo was given three times weekly for 12 weeks to 30 HIV-infected patients that had been treated with HAART for at least 24 weeks and not yet achieved CD4 counts above 350 CD4+ cells/microl. Blood samples were collected at weeks 0, 2, 4, 8, and 12, and again 12 weeks after termination of the G-CSF treatment. Significant increase in absolute numbers of circulating CD34+ cells was detected in the treatment group (P = 0.006). The function of progenitor cells was examined in vitro using a colony-forming unit (CFU) assay, and increase in the number of CFU/ml was detected (P = 0.005). In order to estimate the effect of G-CSF on in vivo function of progenitors the white-blood count was determined. Significant increase in white-blood count was found (P < 0.001), while hemoglobin and platelet count decreased (P = 0.001 and P = 0.013, respectively). Significant increase in the CD4 count occurred, but correlation between the numbers of progenitors and the CD4 count was not found. These data suggest that G-CSF mainly increases the number and differentiation of myeloid progenitors.

Lipopolysaccharide tolerance in relation to intrabronchial influx of neutrophils in the rat.

Lipopolysaccharide (LPS) is a potent chemotactic component for polymorphonuclear leukocytes (PMN, neutrophils). Since LPS tolerance was first described, many studies have been reported about the hyporesponsiveness in vitro corresponding to attenuating production of proinflammatory cytokines. We hypothesized that in vivo daily exposure to LPS stimuli impairs neutrophil accumulation in the rat airway. Interleukin 8 (IL-8) and/or CXC-chemokine, a neutrophil chemoattractant and activating cytokine, have been implicated as proinflammatory mediators in gram-negative respiratory tract infections. It is possible that the tolerance to LPS has occurred in relation to this chemoattractant cytokine production. To settle this issue, we examined whether the neutrophil count in bronchoalveolar lavage fluid (BALF) decreases after daily inhalation of Pseudomonas aeruginosa LPS into the rat airway. Repeated inhalation of LPS into the airway resulted in reduction in neutrophil recruitment. We measured rat CXC-chemokine (rat GRO/CINC1) levels in recovered BALF. There were noted reductions of rat GRO corresponding to the diminished neutrophil trafficking. We also confirmed that the HLA-DR positive lymphocyte number in BALF gradually increased after daily inhalation of LPS. These results suggest that continuous stimuli of LPS mitigate the accumulation of inflammatory cells in the airway by reducing chemokine production with a consequent change in the appearance of local inflammation to a chronic state.

Effects of n-acetylcysteine in a rat model of ischemia and reperfusion injury.

OBJECTIVE: Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of n-acetylcysteine (NAC), a free radical scavenger, in rats subjected to SAO shock. METHODS AND RESULTS: Treatment of rats with NAC (applied at 20 mg/kg, 5 min prior to reperfusion, followed by an infusion of 20 mg/kg/h) attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. NAC also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine in the plasma of the SAO-shocked rats after reperfusion. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) synthetase (PARS) revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and PARS were markedly reduced in tissue sections obtained from SAO-shocked rats which had received NAC. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin, which was mainly localised in the vascular endothelial cells. Ileum tissue section obtained from SAO-shocked rats with anti-intercellular adhesion molecule (ICAM-1) antibody showed a diffuse staining. NAC treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue section from SAO-shocked rats. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). NAC treatment improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and significantly improved survival. CONCLUSION: Taken together, our results clearly demonstrate that NAC treatment exert a protective effect and part of this effect may be due to inhibition of the expression of adhesion molecule and peroxynitrite-related pathways and subsequent reduction of neutrophil-mediated cellular injury.

LSP1 modulates leukocyte populations in resting and inflamed peritoneum.

Lymphocyte-specific protein 1, recently renamed leukocyte-specific protein 1 (LSP1), is an F-actin binding protein expressed in lymphocytes, macrophages, and neutrophils in mice and humans. This study examines LSP1-deficient (Lsp1(-/-)) mice for the development of myeloid and lymphocytic cell populations and their response to the development of peritonitis induced by thioglycollate (TG) and to a T-dependent antigen. Lsp1(-/-) mice exhibit significantly higher levels of resident macrophages in the peritoneum compared to wild-type (wt) mice, whereas the development of myeloid cells is normal. This increase, which is specific for conventional CD5(-) macrophages appears to be tissue specific and does not result from differences in adhesion to the peritoneal mesothelium. The level of peritoneal lymphocytes is decreased in Lsp1(-/-) mice without affecting a particular lymphocytic subset. The proportions of precursor and mature lymphocytes in the central and peripheral tissues of Lsp1(-/-) mice are similar to those of wt mice and Lsp1(-/-) mice mount a normal response to the T-dependent antigen, ovalbumin (OVA). On injection of TG, the Lsp1(-/-) mice exhibit an accelerated kinetics of changes in peritoneal macrophage and neutrophil numbers as compared to wt including increased influx of these cells. LSP1(-) neutrophils demonstrate an enhanced chemotactic response in vitro to N-formyl methionyl-leucyl-phenylalanine (FMLP) and to the C-X-C chemokine, KC, indicating that their enhanced influx into the peritoneum may be a result of increased motility. Our data demonstrate that LSP1 is a negative regulator of neutrophil chemotaxis. (Blood. 2000;96:1827-1835)

A single oral dose of thalidomide enhances the capacity of lymphocytes to secrete gamma interferon in healthy humans.

Thalidomide is increasingly being used as adjuvant therapy for patients with mycobacterial and human immunodeficiency virus (HIV) infections. The T-helper (Th)1 cytokine-Th2 cytokine balance critically determines the outcomes of these diseases. To obtain insight into the effect of thalidomide on the capacity of lymphocytes to produce Th1 and Th2 cytokines, six healthy volunteers received an oral dose (400 mg) of thalidomide. Before and at 3, 6, and 24 h after ingestion of thalidomide, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated for 24 h with the T-cell stimulant staphylococcal enterotoxin B (SEB) or anti-CD3/CD28. In all six volunteers ingestion of thalidomide was associated with enhanced SEB- and anti-CD3/CD28-induced production of the Th1 cytokine gamma interferon (P < 0.05) and a decrease in the level of anti-CD3/CD28-induced interleukin-5 (IL-5) production (P < 0.05). The levels of IL-2 (Th1) and IL-4 (Th2) released remained unchanged. These changes were accompanied by an increase in the amount of IL-12p40 released by the PBMCs 6 h after ingestion of thalidomide (P < 0.05). Thus, a single oral dose of thalidomide causes a Th1-type response in healthy humans. This finding offers a potential explanation for the positive effect of thalidomide in patients with mycobacterial and HIV infections.

Lenograstim as support for ACE chemotherapy of small-cell lung cancer: a phase III, multicenter, randomized study.

This phase III study was conducted to evaluate the usefulness of lenograstim as support for ACE (doxorubicin, cyclophosphamide, and etoposide) chemotherapy in previously untreated patients with small-cell lung cancer. Patients were randomized to receive up to six 3-week cycles of either ACE alone (n = 139) or ACE with lenograstim support (150 microg/m2/day subcutaneously, days 4-13, n = 141). Compared with the chemotherapy-alone group, the lenograstim support group was more likely to achieve neutrophil recovery (absolute neutrophil count, > or =1.5 x 10(9) cells/l) by day 14 (95.8-100% vs. 14.3-24.1% across the cycles) and less likely to experience at least one infectious episode (36.7 vs. 54.0%; p = 0.004), chemotherapy delay (51.8 vs. 56.2%; NS), or dose reduction (17.3 vs. 27.7%; p = 0.037). Objective response and event-free and overall survival rates were similar. Lenograstim was well tolerated. Lenograstim may allow the interval between cycles of ACE to be reduced to 2 weeks; such dose intensification may lead to more favorable objective response and survival rates.

Interactions between blood cells and retinal endothelium in endotoxic sepsis.

Platelets and leukocytes are thought to play a leading role in the pathogenesis of many inflammatory conditions. To recruit flowing blood cells to the inflammatory region, it would be necessary for them to interact with vascular endothelial cells. Recently, many reports have indicated the resistance of spontaneous hypertensive rats (SHR) to endotoxic sepsis. Their resistance might be derived from suppressed interaction between these blood cells and endothelial cells. Therefore, SHR and age-matched Wistar-Kyoto rats (WKY) were induced with endotoxic sepsis by intravenous injection of lipopolysaccharide (LPS). At 4, 12, 24, and 48 hours after induction, leukocyte-endothelial interactions in the retina were evaluated in vivo with acridine orange digital fluorography. Fluorescently labeled platelets were also injected to investigate platelet-endothelial interactions in the retina in endotoxic sepsis. Leukocyte rolling in SHR after LPS injection was significantly suppressed; the maximum number of rolling leukocytes was reduced by 80.1% at 12 hours after LPS injection in SHR compared with WKY. Subsequent leukocyte infiltration into the vitreous cavity was significantly inhibited in SHR. Furthermore, platelet-endothelial interactions in the retina were also suppressed in SHR treated with LPS. The maximum numbers of rolling and adherent platelets were reduced by 59.5% and 62.6%, respectively, in SHR compared with WKY. In both strains, leukocyte- and platelet-endothelial interactions were substantially inhibited by the blocking of P-selectin. These suppressed interactions could contribute to the reduction of leukocyte- and platelet-mediated tissue injury in endotoxic sepsis in SHR, resulting in their resistance to endotoxemia.

Topical steroid treatment of allergic rhinitis decreases nasal fluid TH2 cytokines, eosinophils, eosinophil cationic protein, and IgE but has no significant effect on IFN-gamma, IL-1beta, TNF-alpha, or neutrophils.

BACKGROUND: Topical treatment with glucocorticoids (GCs) is known to decrease eosinophils but not neutrophils in patients with allergic rhinitis. OBJECTIVE: We sought to examine whether the differential effects of GC treatment on eosinophils and neutrophils are mirrored by differential effects on T(H)1/T(H)2 cytokines and the neutrophil-associated cytokines IL-1beta and TNF-alpha. METHODS: Differential counts of eosinophils and neutrophils in nasal fluids from 60 children with seasonal allergic rhinitis treated with a topical GC were examined after staining with May-Grünwald-Giemsa stain. Nasal fluid levels of IFN-gamma, IL-4, IL-6, IL-10, IL-1beta, and TNF-alpha were examined with ELISA, and IgE and eosinophil cationic protein (ECP) levels were examined with RIA. RESULTS: After GC treatment, there was a statistically significant decrease of the T(H)2 cytokines IL-4, IL-6, and IL-10, as well as ECP and IgE. By contrast, there were no significant changes of the levels of IFN-gamma, IL-1beta, TNF-alpha, or neutrophils. In the GC-treated patients IL-1beta and TNF-alpha levels correlated with neutrophils and ECP, and IL-1beta correlated with eosinophils. Furthermore, ECP correlated with both eosinophils and neutrophils. Neither IL-1beta nor TNF-alpha correlated with IgE. Patients with high neutrophil counts after GC treatment were found to have significantly higher eosinophil counts and ECP than patients with low counts. CONCLUSIONS: The beneficial effects of topical treatment with GC in patients with allergic rhinitis could be attributed to downregulation of T(H)2 cytokines, with an ensuing decrease of eosinophils, ECP, and IgE. It is possible that neutrophils could counteract the beneficial effects of GCs by releasing the proinflammatory cytokines IL-1beta and TNF-alpha.

Haematological toxicity following different dosing schedules of 5-fluorouracil and epirubicin in rats.

AIM: To study the effects of single and fractionated doses of 5-fluorouracil and epirubicin on the leukocyte counts in rats. METHODS: Six different dosing patterns of each drug were injected within one day. The leukocytes were followed for 11-15 days. Pharmacokinetic models were developed using NONMEM. Quantitative and qualitative pharmacokinetic-pharmacodynamic relationships were investigated. RESULTS: A one-compartment model with non-linear elimination described 5-fluorouracil pharmacokinetics and a three-compartment model described epirubicin concentration data. Sigmoidal or basic Emax-models quantified the relationships between individual AUCs and decreases in leukocytes, for both drugs. Similar relationships between AUC and toxicity were found, regardless of whether the drugs were given as single or fractionated doses. CONCLUSION: Quantitative relationships between AUC and the effect on leukocytes were established for 5-fluorouracil and epirubicin. However, no schedule dependence was indicated for the schedules used in the study.

[Do hematopoietic growth factors have a role in the treatment of drug-induced agranulocytosis?]. / Les facteurs de croissance hématopoïétique ont-ils une place dans le traitement des agranulocytoses médicamenteuses?

PURPOSE: Our study was aimed at determining the role of hematopoietic growth factor in drug-induced agranulocytosis. METHODS: Fifty-five cases of drug-induced agranulocytosis were reviewed and subdivided retrospectively into a G-CSF group (n = 15) and an untreated group (n = 40). Mortality and hematological recovery (number of days required for neutrophil counts to exceed 1.5 x 10(9)/L) were studied in the two groups. RESULTS: The mean granulocyte count was 0.09 x 10(9)/L. All patients presented infection. In the G-CSF group, no mortality (0% versus 5%, P = 0.85) and a shorter recovery time (8.1 versus 9.5 days P = 0.39) were observed. No significant difference between the two groups was observed in either the duration of antibiotic therapy (9.3 days in the G-CSF group versus 10.1 days in the untreated group, P = 0.51) or duration of hospitalization (10 days in the treated group versus 11 days in the G-CSF group, P = 0.46). CONCLUSION: Our results as well as a literature review indicate that G-CSF could decrease the time to hematological recovery and perhaps reduce mortality. However, the exact role of hematopoietic growth factors requires further investigations.

Randomized, dose-escalation study of SD/01 compared with daily filgrastim in patients receiving chemotherapy.

PURPOSE: To explore the use of SD/01 (a polyethylene glycol-conjugated filgrastim shown in preclinical studies to have a prolonged half-life) in patients with chemotherapy-induced neutropenia. PATIENTS AND METHODS: Thirteen patients with non-small-cell lung cancer were randomized to receive daily filgrastim (5 microg/kg/d) or a single injection of SD/01 (30, 100, or 300 microg/kg) 2 weeks before chemotherapy and again 24 hours after administration of carboplatin and paclitaxel. Pharmacodynamic, pharmacokinetic, and safety analyses were performed. RESULTS: Peak serum concentrations of SD/01 and the duration of increased serum concentrations were dependent on the SD/01 dose. SD/01 concentrations remained increased longer in patients with chemotherapy-induced neutropenia. Prechemotherapy median absolute neutrophil counts (ANCs) in patients receiving SD/01 were increased in a dose-dependent fashion, with the duration of this effect also being dose dependent. After chemotherapy, median ANC nadirs were similar in the filgrastim cohort and the cohort receiving SD/01 30 microg/kg, with higher nadirs seen in the cohorts receiving SD/01 100 or 300 microg/kg. Dose-limiting toxicities were not noted. CD34(+) cells were mobilized in all cohorts. CONCLUSION: A single dose of SD/01 increases the serum concentration of SD/01 for several days in a dose-dependent fashion and is not associated with significant toxicity. The effects of SD/01 on ANC and CD34(+) cell mobilization are comparable or greater than those achieved with daily filgrastim. The self-regulation of this molecule provides a potential therapeutic advantage in a variety of clinical settings associated with neutropenia.

Immunological effects of oral high-dose methylprednisolone in acute optic neuritis and multiple sclerosis.

The immunological effects of high-dose methylprednisolone in attacks of multiple sclerosis and acute optic neuritis have only been examined in a few randomized, controlled trials. We studied immunological changes in 50 patients with optic neuritis or multiple sclerosis who underwent lumbar puncture before and 1 week after completing a 15-day course of oral high-dose methylprednisolone treatment. Treatment resulted in a decrease in the concentration of myelin basic protein, a decrease in the serum concentration of immunoglobulin G (IgG) and intrathecal IgG synthesis, an increase in the cerebrospinal fluid concentration of transforming growth factor-beta1, and changes in the expression of CD25, CD26, and human leukocyte antigen-DR (HLA-DR) on CD4 T-cells. No effect was seen on the cerebrospinal fluid leucocyte count or the cerebrospinal fluid activity of matrix metalloproteinase-9 (MMP-9). The lack of a persistent effect on cerebrospinal fluid leucocyte recruitment and MMP-9 activity, despite changes in IgG synthesis, T-cell activation, and cytokine production, suggests that modulation of the function of inflammatory cells may contribute to the clinical efficacy of oral high-dose methylprednisolone treatment in optic neuritis and multiple sclerosis.