Granulocyte Suspensions Research Articles

Granulocyte Transfusions in Neutropenic Infections: Insights From a Single-Center Study.

Introduction Despite the development of modern antibiotic and antifungal therapies, neutropenic infections remain life-threatening. Granulocyte transfusion (GTX) is a less frequently used treatment modality in patients with refractory neutropenic infections. The role of donor GTX remains controversial, partly because of the lack of proper clinical trials. This study aimed to contribute to the literature by evaluating the efficacy and side effects of granulocyte transfusions in our center. Methods Eight febrile neutropenic patients with confirmed infections received granulocyte transfusions from ABO-compatible related and unrelated donors. Donors received filgrastim and dexamethasone stimulation, and granulocyte suspensions were irradiated and administered within six hours. Monitoring, antibiotic therapy, and granulocyte colony-stimulating factor (G-CSF) support were maintained. Results Our study observed a 28-day survival rate of 25%, which was lower than that reported in previous literature. The median number of transfusions was 3, with an average eight-day duration post-infection diagnosis, and no side effects were observed. Conclusion While some patients benefited from GTX, overall survival rates remained modest, indicating the need for further research. Prospective, well-powered randomized controlled trials are essential to address patient selection, dosing, and duration to determine the clinical utility of GTX. This study underscores the complexity of GTX in real-world clinical practice and provides insight into the ongoing debate regarding its efficacy in treating severe neutropenic infections.

БИОФИЗИЧЕСКИЕ СВОЙСТВА КЛЕТОЧНОЙ ПОВЕРХНОСТИ И ФУНКЦИОНАЛЬНАЯ АКТИВНОСТЬ ГРАНУЛОЦИТОВ БОЛЬНЫХ ОСТРЫМ ЛИМФОБЛАСТНЫМ ЛЕЙКОЗОМ ПРИ МОДЕЛИРОВАНИИ НАГРУЗКИ С АТФ IN VITRO

The current study is devoted to the investigation of the biophysical properties of the cell surface and the functional activity of granulocytes in patients with acute lymphoblastic leukemia when simulating the ATP load in vitro. The experiment was performed on the peripheral blood of patients with acute lymphoblastic leukemia (ALL) who underwent a standard course of chemotherapy. In experimental tests, exogenous loading with ATP was simulated in vitro by adding 100.0 μM adenosine-5-triphosphate disodium salt trihydrate to the granulocyte suspension. Incubation with the drug was carried out for 15 min at 370 C. As a control, a suspension of granulocytes in RPMI 1640 medium from the same patient, but without the addition of the drug, was used, this was incubated for 15 min at a temperature of 370 C. After the incubation time, the biophysical properties (rigidity, charge of the cell surface, the strength of intercellular adhesion) of granulocytes in experimental and control samples were studied using atomic force microscopy, and the migration activity of cells was also assessed in a direct capillary test under agarose. A model with exogenous ATP in ALL patients showed a decrease in the rigidity and potential of the plasma membrane surface, an increase in the adhesive properties and migration activity of granulocytes. The revealed effects point to the key role of the ATP molecule in the mechanisms of intercellular signaling in the microvasculature.

Efficacy and safety of granulocyte transfusion in children: A single‐center experience

Granulocyte suspension transfusion (GTx) can be used in severely neutropenic patients with infections that cannot be controlled despite appropriate antibiotic therapy. We aimed to evaluate the effectiveness and safety of GTx for the treatment of febrile neutropenia (FEN) in the pediatric age group. Patients who underwent GTx in the Hematology Clinic of Ankara Child Health and Diseases Hematology Oncology Training and Research Hospital between 2013 and 2017 were evaluated retrospectively. Hematologic and clinical response rates, effects on survival, and adverse effects were investigated. Clinical response was defined at two time points: clinical response I was evaluated after each transfusion, while clinical response II was evaluated after the final GTx in a FEN episode. During the study period, 343 GTx were given 107 FEN episodes of 74 patients. The mean number of granulocyte suspensions administered per patient and per FEN episode was 4.6 units and 3.2 units. The mean GTx volume administered was 237 ± 40 mL, and the mean granulocyte count was 2.8 ± 1.3 x 1010 /unit. Hematologic response was attained in 163 (47.6%) of 343 transfusions. Clinical response I was obtained in 88 (25.7%) of the GTx, and clinical response II was attained in 83 (78.5%) of 107 episodes. Life-threatening adverse event was not observed. The cumulative 1-month and 3-month survival rates were 87.8% and 76.5%, respectively. High hematologic response and clinical recovery rates were achieved with GTx, with no limiting adverse effects. Granulocyte transfusion appears to be a safe and effective treatment in pediatric patients with FEN.

Role of rhIL-17 in regulating the mitochondrial pathway proteins in peripheral blood neutrophils.

IL-17, classified as an inflammatory cytokine, plays a key role in the activation of inflammatory processes involving neutrophils. Twenty healthy voluntary blood donors were controlled in the study. The granulocyte suspensions were stimulated with rhIL-17 and fMLP. Expression of Bcl-xl, Smac/DIABLO, and Omi/HtrA2 in neutrophil lysates were assessed by Western blot. The level of cytochrome c and activity of caspase 9 was also assayed in these cells. The results of existing research highlight the importance of rhIL-17 in reducing the survival of neutrophils via the mitochondria, depending on the Bcl-2 protein family. Our research has indicated that rhIL-17 regulates the mutual relationships between the proteins of that family. The proapoptotic effect observed in neutrophils affected by rhIL-17 is a result of a decreased expression of Bcl-xl. Consequently, the expression of apoptogenic proteins, including cytochrome c, Smac/DIABLO, and Omi/HtrA2, is elevated. Surprisingly, there have been no observations of the cytokine influencing the activity of caspase 9. Results have shown for the first time that IL-17 has a direct effect on the decrease of Bcl-xl. In conclusion, the results of the research presented in this article confirm the dual action of IL-17, which, on the one hand, leads to an array of proinflammatory mechanisms regarding neutrophils and, on the other hand, reduces the survival of those cells via an immediate influence on the Bcl-2 family of proteins and apoptogenic factors.

The effect of estrogens on superoxide anion generation by human neutrophil granulocytes: Possible consequences of the antioxidant defense

The present study aimed to test whether, beyond the known antioxidant effect of estradiol, such a property is also possessed by estrone and estriol. For this purpose, an in vitro investigation of the effect of estrone and estriol on superoxide anion production by human neutrophil granulocytes was carried out. Blood samples were obtained from healthy volunteers and neutrophil granulocytes were separated for measurement of superoxide anion generation after incubation with estrone, estriol (10−7, 10−6 and 10−5 M) and 17β-estradiol (10−7 M). Superoxide anion production of isolated neutrophil granulocytes was quantified by photometry and using the reduction of ferricytochrome-C. When adding estrone and estriol to neutrophil granulocyte suspensions, the production of superoxide anion fell (10−5 M: 84.17 ± 3.14% and 88.77 ± 1.98% of control production, p < 0.01 and p < 0.05, respectively). Estradiol produced an antioxidant effect at lower concentration (10−7 M: 72.91 ± 7.94% of control production, p < 0.001). The weak estrogens estrone and estriol, similarly to estradiol, are also able to reduce the superoxide anion release in our experimental model. This may have importance in the antioxidant defense of biological systems.

Inhibition of 5-lipoxygenase and Leukotriene C4 Synthase in Human Blood Cells by Thymoquinone

Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 μM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 μM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 μM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.

Induced myeloperoxidase activity and related superoxide inhibition during hormone replacement therapy

Objective To test whether the menopause entails any changes in the myeloperoxidase activity of neutrophil granulocytes. The effects of hormone replacement therapy on myeloperoxidase activity and related changes in free radical production were also investigated. Design Laboratory investigation of the effect of oestrogen on intracellular myeloperoxidase activity and release from human neutrophil granulocytes. Analysis of related changes in superoxide anion generation. Setting 2nd Department of Medicine and 1st Department of Obstetrics and Gynaecology, Semmelweis University, Budapest. Samples Intracellular myeloperoxidase activity (mean peroxidase index) was measured automatically in blood samples obtained for general laboratory work-up from 135 randomly selected patients in our department. Blood samples from 11 postmenopausal women were analysed before and during hormone replacement therapy. Blood samples from 20 healthy volunteers were obtained and neutrophil granulocytes separated for in vitro measurement of superoxide anion production after adding myeloperoxidase to the incubation media. Methods The mean peroxidase index was measured using a Technicon H-3 instrument. myeloperoxidase release from neutrophils was quantified by ELISA technique. Superoxide production of isolated neutrophil granulocytes was measured by photometry. Main outcome measures Intracellular activity of myeloperoxidase, concentration of myeloperoxidase-protein in supernatant of neutrophils, release of superoxide anion from neutrophil granulocytes. Results 1. Intracellular myeloperoxidase activity in neutrophils was lower in postmenopausal women, than in females with regular cycles (-1.84 ±3.06 versus 1.59 ±3.55, P<0,001). 2. In postmenopausal women intracellular myeloperoxidase activity and myeloperoxidase release increased during hormone replacement therapy (-5.54 ±6.63 versus -0.2 ±6.05; P<0.001 and 52.74 mU/ml ±25.73 versus 251.4 ±234.1 mU/ml; P<0.05). 3. Adding myeloperoxidase to neutrophil granulocyte suspensions, the production of superoxide anion fell (e.g. adding 280 ng/ml myeloperoxidase: 77.9 ±14.04 % of control production, P<0.001). Conclusion Hormone replacement restores the reduced myeloperoxidase activity in menopausal women. Adding myeloperoxidase to neutrophil granulocytes, the production of free radicals decreases.

Impaired leukotriene C4 generation in granulocytes from protein-energy malnourished chronically ill elderly.

The dysregulation of the immune and inflammatory systems observed in protein-energy malnutrition (PEM) may be partly due to perturbation of essential fatty acid metabolism. In this study, we assessed the calcium ionophore A23187-induced generation of the arachidonate metabolites leukotriene B4 (LTB4) and leukotriene C4 (LTC4) in isolated granulocyte suspensions. Case-control study. A university-affiliated acute care hospital in urban Stockholm. Fourteen severely malnourished elderly subjects with stable non-malignant disorders (age 74 +/- 1 years, mean +/- SEM) and 12 healthy age-matched controls were examined. Leukotrienes were analysed by high-performance liquid chromatography. Body mass index (BMI, kg m-2) and delayed cutaneous hypersensitivity (DCH) reaction were determined. BMI was 16. 5 +/- 0.5 and 26.2 +/- 0.9 kg m-2 (mean +/- SE) in the malnourished group and controls (P < 0.001), respectively. DCH was 8.5 mm (median) in patients and 29.5 mm in controls (P < 0.001). LTC4 generation in granulocytes from PEM patients was half of that of controls (9.1 +/- 2.0 vs. 17.8 +/- 5.2 pmol mL-1, P < 0.05) when cells were stimulated with 0.2 micromol L-1 of A23187, and 13.7 +/- 2.5 and 27.2 +/- 7.5 pmol mL-1, respectively (NS), upon stimulation with 1.0 micromol L-1 of A23187. LTB4 production in PEM patients and controls did not differ at any of the two calcium ionophore concentrations. LTC4 production correlated with BMI (r = 0.41, P < 0.05), but there was no significant correlation between DCH and LTB4 or LTC4 production. Protein-energy malnutrition is accompanied by perturbation of leukotriene synthesis, which may be one factor underlying the dysregulation of inflammatory responses in the depleted patient.

Activation of human granulocytes by intravenous immunoglobulin preparations is mediated by FcgammaRII and FcgammaRIII receptors.

Previous studies from our laboratory have shown that i.v. Ig (IVIG) exposure triggers superoxide anion (O2) generation by and increases bactericidal capacity of human blood granulocytes. However, the molecular interactions between IVIG and granulocytes have not been evaluated before. The objective of this study was to investigate the role of FcgammaRII and FcgammaRIII receptors in the immunomodulatory effects of IVIG concentrates on granulocytes. We found that four different IVIG preparations (concentration range, 1-10 mg/mL) shared the ability to stimulate O2- release in vitro by granulocytes in a dose-dependent manner. Dimers fractionated from IVIG were significantly more potent in inducing activity of the respiratory burst than were monomers. MAb (concentration range, 0.1-10 microg/mL) specific for FcgammaRII and FcgammaRIII receptors inhibited the IVIG-induced O2- release, with a more profound inhibitory effect observed with anti-FcgammaRIII. These findings suggest the involvement of Fcgamma receptors in triggering O2- release by granulocytes exposed to IVIG. We also report that IVIG added to granulocyte suspensions elicited a rapid and vigorous increase in the concentration of cytosolic free calcium, a finding suggesting direct activation and not priming of granulocytes by IVIG through FcgammaRII and FcgammaRIII receptors. The in vitro effects described here might occur in patients treated with IVIG and may, in part, be responsible for inflammatory reactions evoked by infused Ig concentrates as well as the immunomodulatory effect of Ig in patients with autoimmune and inflammatory diseases.

Aberrant expression of active leukotriene C4 synthase in CD16+ neutrophils from patients with chronic myeloid leukemia

Elevated leukotriene (LT)C(4) synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16(+) neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16(+) neutrophils from all tested patients transformed exogenous LTA(4) to LTC(4). These cells also produced LTC(4 )after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC(4) synthase mRNA in CML CD16(+) neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC(4) synthase at the protein level in CML CD16(+) neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC(4) synthase activity or expression of the protein could not be demonstrated in CD16(+) neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16(+) neutrophils, transformed LTA(4) to LTB(4). The results indicate that aberrant expression of LTC(4) synthase is a regular feature of morphologically mature CML CD16(+) neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC(4) synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC(4 )has been demonstrated to stimulate proliferation of human bone marrow-derived myeloid progenitor cells. (Blood. 2000;95:1456-1464)

High oxygen radical production is associated with fast parasite clearance in children with Plasmodium falciparum malaria.

It has been hypothesized that reactive oxygen intermediates (ROI) released by leukocytes play a major role in the immune response to many infectious agents. In the present study, the parasitologic and clinical courses of 75 Gabonese children with Plasmodium falciparum malaria were compared with the ability of their granulocytes to produce oxygen radicals. The luminol-dependent chemiluminescence in granulocyte suspensions for the children was measured without stimulation and after stimulation with phorbol-12-myristate-13-acetate, N-formyl-methionyl-leucyl-phenylalanine, or tumor necrosis factor. A significant association was found between fast parasite clearance time and high oxygen radical generation in both the unstimulated and stimulated granulocyte preparations. No correlation was found between fever clearance time and ROI generation. These findings suggest that ROI play a pivotal role in the immune response as a first line of defense against P. falciparum malaria.

Differences in the anti-inflammatory effects of theophylline and pentoxifylline: important for the development of asthma therapy?

Antiasthma drugs are now being re-evaluated for their anti-inflammatory effects. Theophylline is an immunomodulator; however, weak effects and the narrow therapeutic window make it a controversial drug. We compared the immunomodulatory potencies of theophylline with those of the xanthines pentoxifylline (POF) and A802715. Using a whole-blood, cell-culture system, we studied the effects on the release of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin-6 (IL-6) in six healthy subjects, and, in granulocyte suspensions, the effects on the release of reactive oxygen species (ROS). We also studied the influence of a 14-day treatment with theophylline or POF on the release of the cytokines named above in 14 asthmatics. We found that equimolar concentrations of A802715 most effectively inhibit ROS generation, followed by POF; the effects of theophylline were weakest. A802715-inhibited release of TNF-alpha was four times as potent as that of theophylline, and POF two times as potent. Inhibition of IFN-gamma by A802715 was three times as potent, and by POF two times. Neither drug influenced IL-6 release. After a 14-day treatment of asthmatics, POF proved to inhibit TNF-alpha release more effectively (by 44.3%) than theophylline (7.5%). It is concluded that study of xanthine derivatives in asthmatics might help the development of asthma therapy. POF seems to be an especially promising candidate.

Evaluation of neutrophilic function (chemotaxis, phagocytosis and microbicidal activity) in healthy dogs and in dogs suffering from recurrent deep pyoderma

The modified Boyden's technique of chemotaxis (`leading front' method) and the bacterial killing test with Staphylococcus sp. stained with acridine orange were performed with suspensions of granulocytes from 50 healthy dogs. Lipopolisacharide of Escherichia coli, in normal dog serum was used as the chemotactic factor. The mean value for the chemotactic differential found was 30.41±12.14 μ. The optimal concentration of bacteria and opsonins (normal dog serum) was 4 and 15%, respectively, and the ideal incubation period was 30 min. The mean values obtained for phagocytosis percentage, number of bacteria per PMN, phagocytosis index and percentage of dead bacteria, were 97.01±3.22%, 22.20±4.6, 21.53±4.50 and 45.30±9.18%, respectively. Granulocyte functions in 18 dogs with recurrent, chronic, deep pyoderma were assessed by the same methods. No significant difference between the two groups, for any of the evaluated parameters, was found. For elucidation of the pathogenesis of recurrent pyoderma, further studies involving humoral and cellular immunity and the complement system are required.

Ionophore A23187-induced leukotriene biosynthesis in equine granulocytes—neutrophils, but not eosinophils require exogenous arachidonic acid

Equine granulocyte suspensions, mainly consisting of neutrophils, failed to produce detectable amounts of leukotrienes when stimulated with calcium ionophore A23187 alone, whereas leukotrienes were dose-dependently formed in control incubations with human granulocytes. In contrast, ionophore A23187 initiated synthesis of leukotrienes B 4 and C 4 in equine granulocytes when added in combination with low concentrations of exogenous arachidonic acid. Similarly, ionophore A23187 provoked leukotriene biosynthesis when added alone to human whole blood, whereas addition of exogenous arachidonic acid was a prerequisite for ionophore A23187-induced leukotriene formation in equine whole blood. Leukotriene biosynthesis was provoked by A23187 alone after addition of homologous platelets to equine granulocyte suspensions. After separation of equine neutrophils and eosinophils, purified eosinophil suspensions produced LTC 4 after stimulation with ionophore A23187 alone, whereas exogenous arachidonic acid was required for ionophore-induced LTB 4 formation in purified neutrophil suspensions. Leukotriene synthesis in both eosinophils and neutrophils was suppressed by the 5-lipoxygenase activating protein (FLAP) inhibitor, MK-886. Exogenous arachidonic acid was needed for ionophore-induced leukotriene synthesis also in bovine granulocytes, but was not a prerequisite for the production of leukotrienes in porcine granulocytes or in rat and rabbit white blood cell suspensions. The results indicate differences in the mechanisms regulating leukotriene synthesis in equine neutrophils, as compared to human granulocytes or equine eosinophils, and suggest that elevation of intracellular calcium is an insufficient stimulus to provoke utilisation of endogenous arachidonic acid for leukotriene synthesis in equine neutrophils.

Biomaterial-dependent blood activation during simulated extracorporeal circulation: a study of heparin-coated and uncoated circuits.

Blood activation during extracorporeal circulation is associated with morbidity and mortality in cardiac surgery. This activation can be diminished by usage of heparin-coated circuits. Nitric oxide has also been reported to influence humoral and cellular components of blood. This study was performed to determine biomaterial-dependent part of blood activation. Fresh, whole human blood mixed with Ringer's solution was circulated through a heart-lung machine for two and half hours. Five circuits were heparin-coated (group HC), whilst five other circuits were uncoated (group NC). During the last half hour NO was added to the oxygen/air mixture. Blood activation was estimated by measuring following parameters: interluekin 6, complement activation products C3a and terminal complement complex, and oxygen free radicals (OFR) production capacity, which was determined using chemiluminescence enhanced by serum opsonized zymosan (SOZ) and phorbol myristate acetate (PMA). Granulocyte activation was measured as release of myeloperoxidase (MPO) and human neutrophil lipocalin (HNL). OFR in granulocyte suspension stimulated by SOZ and PMA were significantly lower in the NC group, mostly later during ECC. Similarly, lower neutrophil and monocyte counts were observed in this group. NO increased superoxide production in the whole blood in heparin-coated circuits, but did not change OFR in isolated granulocytes. MPO was also affected by heparin-coating. NO supply seemed to increase release of MPO and HNL. It is concluded that heparin-coating contributed to reduction of biomaterial-dependent blood activation. An addition of NO at late stage of ECC tended to influence this activation.

Exposure of blood to biomaterial surfaces liberates substances that activate polymorphonuclear granulocytes

Human whole blood, anticoagulated or not, was exposed to hydrophilic glass surfaces or methylated hydrophobic glass surfaces under saline cover. Platelet-poor plasma or serum was prepared after 10 minutes of exposure, measured in respect to complement activation, and transferred to a suspension of granulocytes, which acted as bioprobes. The granulocytes were prepared from blood, anticoagulated with ethylenediaminetetraacetic acid, and evaluated regarding intracellular Ca 2+ concentration (Calcium Green-1 fluorescence), integrin expression (CD-11b immunohistochemistry), respiratory burst (chemiluminescence), and priming (increase in N-formyl-methionyl-leucyl-phenylalanine-induced respiratory burst). The results indicate that humoral factors formed during the surface exposure of blood were able to activate the probe granulocytes. The exposure to hydrophilic surfaces led to a calcium transient three times the magnitude of that of hydrophobic surfaces. This response could be blocked by the presence of heparin during the blood-surface exposure but was not affected by the addition of heparin to the probe granulocytes. Hirudin, a specific thrombin blocker, had no effect. The exposure to hydrophobic surfaces led to complement activation in serum that induced priming and respiratory burst of the probe granulocytes. In conclusion, the study provides evidence that hydrophilic-hydrophobic surface treatment significantly affects the immediate inflammatory response of a blood-biomaterial interaction that is moderated by the presence of heparin.

Cytotoxicity of magnetite-loaded polylactide, polylactide/glycolide particles and solid lipid nanoparticles

Particles from polylactide (PLA), polylactide/glycolide (PLA/GA) and solid lipid nanoparticles (SLN) were produced and loaded with different amounts of magnetite. The in vitro cytotoxicity was determined to assess their toxicological acceptance as intravenous formulation for magnetic resonance imaging and as potential carrier for drug targeting. Viability determinations were performed in suspensions of human granulocytes using the dimethylthiazolyldiphenyltetrazolium (MTT) test. Particle internalization by the granulocytes was followed using luminol enhanced chemiluminescence (CL). The effective concentrations to reduce the viability to 50% (ED 50%) were 0.38% and 0.30% for high and low molecular weight PLA, 0.15% for PLA/GA. The mechanism of toxicity is the intracellular uptake, with the highest toxicity being observed for the faster degrading polymers, low molecular weight PLA and PLA/GA. The solid lipid nanoparticles proved to be the least cytotoxic preparation with an effective concentration (ED 50%) above 10%.

Granulocyte Accumulation in the Transplanted Liver Does Not Correlate with Clinical and Histologic Evidence of Dysfunction

To evaluate the role of granulocytes in reperfusion injury after liver transplantation. The authors injected radiolabeled granulocytes to determine if human liver graft outcome could be correlated with granulocyte accumulation. Pure granulocyte suspension was prepared from eight patients 12 to 24 hours after orthotopic liver transplantation. The granulocytes were labeled with indium-111 (111In) oxine and reinjected. Total body radionuclide images were performed. Liver uptake of granulocytes was compared with biochemical and histologic evidence of liver injury. No correlation was found between liver uptake of granulocytes, as measured by geometric mean counts, and the biochemical or histologic measures of liver injury. Liver uptake of 111In was 9.6% for the patient who had liver dysfunction and 10.4% mean of the study group. This technique did not detect early signs of liver dysfunction. This investigation supports the premise that granulocytes do not play a major role in reperfusion injury of the newly transplanted liver graft.

Interactions of endotoxin with human blood cells and serum proteins.

Endotoxin interacts with several plasma protein systems and blood cells, causing release of a multitude of endogenous mediators that contribute to the pathophysiological process of sepsis. Binding of 125I-labelled lipopolysaccharide, LPS, to human blood in vitro showed that the major part of the 125I-LPS was recovered in plasma, whereas only small amounts were retained in washed suspensions of granulocytes, erythrocytes, monocytes and lymphocytes, respectively. Whole leukocyte preparations or isolated subpopulations incubated with 125I-LPS or fluorescein-conjugated LPS followed by autoradiography, flow cytometry or immunofluorescence microscopy showed unequivocally that monocytes bound much more LPS than did granulocytes and lymphocytes. Lipoprotein electrophoresis followed by autoradiography showed that 125I-LPS bound to all the purified lipoprotein fractions, which was also confirmed by gel filtration chromatography. These findings demonstrate that monocytes represent the most important blood cell for LPS binding and that radiolabelled LPS is able to bind to lipoproteins as well as to other serum constituents.

Phorbol ester-induced suppression of leukotriene C 4 synthase activity in human granulocytes

The effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), on the metabolism of exogenous leukotriene (LT)A 4 in human granulocytes was investigated. After incubation with LTA 4 decreased levels of LTC 4 but not LTB 4 were observed in granulocyte suspensions pretreated with PMA. This finding could in part be ascribed to oxidative metabolism of LTC 4, since PMA induced a rapid degradation of exogenously added LTC 4. After blocking of LTC 4 metabolism with the H 2O 2 scavenger catalase, a PMA-provoked suppression of the conversion of LTA 4 to LTC 4 was observed, indicating PKC-dependent regulation of LTC 4 synthase activity. This effect, as well as PMA-induced degradation of LTC 4 was presented by specific protein kinase C inhibitors.