Viability Of Granulocytes Research Articles

Effect of silica-based mesoporous nanomaterials on human blood cells

Different mesoporous nanomaterials (MSNs) are constantly being developed for a range of therapeutic purposes, but they invariably interact with blood components and may cause hazardous side effects. Therefore, when designing and developing nanoparticles for biomedical applications, hemocompatibility should be one of the primary goals to assess their toxicity at the cellular level of all blood components.The aim of this study was to evaluate the compatibility of human blood cells (erythrocytes, platelets, and leukocytes) after exposure to silica-based mesoporous nanomaterials that had been manufactured using the sol-gel method, with Ca and Ce as doping elements.The viability of lymphocytes and monocytes was unaffected by the presence of MSNs at any concentration. However, it was found that all nanomaterials, at all concentrations, reduced the viability of granulocytes. P-selectin expression of all MSNs at all concentrations was statistically significantly higher in platelet incubation on the first day of storage (day 1) compared to the control. When incubated with MSNs, preserved platelets displayed higher levels of iROS at all MSNs types and concentrations examined. Ce-containing MSNs presented a slightly better hemocompatibility, although it was also dose dependent.Further research is required to determine how the unique characteristics of MSNs may affect various blood components in order to design safe and effective MSNs for various biomedical applications.

Granulocyte concentrate splitting does not affect phenotype and function

More granulocyte concentrates (GCs) could be produced for more patients from the same donor if apheresis bags were split and stored for longer periods of time. Hence, we tested the hypothesis that splitting and extension of storage of GCs do not impair granulocyte function or viability. Granulocyte apheresis concentrates were produced using modified fluid gelatin as a separation enhancer, split into two portions, and stored for 24 and 48 h. Granulocyte function, represented by cell migration, reactive oxygen species (ROS) production, and neutrophil extracellular trap formation (NETosis), was measured by live-cell imaging. ROS production, adhesive surface protein expression, and viability were measured by flow cytometry. Splitting had no effect on any of the tested parameters. After 24 h of storage, live-cell imaging showed no significant difference in migration, time to maximum ROS production, time to half-maximum NETosis, viability, or CD11b expression, but ROS production induced by phorbol 12-myristate 13-acetate (PMA) decreased from an initial median fluorescence intensity of 1775-590 artificial units. After 48 h, PMA-induced ROS production, viability, and migration declined, as reflected by decreases in median total distance (119 vs. 63.5 μm) and median Euclidean distance (30.75 vs. 14.3 μm). Splitting GC products has no effect on granulocyte viability or function, but extended storage >24 h does compromise granulocyte function. The findings confirm that GCs should be transfused within 24 h of collection. Longer storage cannot be recommended.

Excretory products of the cestode, Schistocephalus solidus, modulate in vitro responses of leukocytes from its specific host, the three-spined stickleback (Gasterosteus aculeatus)

Helminth parasites have evolved remarkable strategies to manipulate the immune system of their hosts. During infections of three-spined stickleback (Gasterosteus aculeatus) with the cestode Schistocephalus solidus prominent immunological changes occur, presumably due to manipulative activity of the parasite. We hypothesise that excretory/secretory products of the parasite are involved in the manipulation of the stickleback's immune system and that this may depend on the individual parasite and its origin. We therefore produced S. solidus conditioned cell culture media (SSCM) with parasites from different origins (Norway, Spain and Germany) and exposed head kidney leukocytes (HKL) from un-infected sticklebacks in cell cultures to SSCM. After in vitro culture, HKL were subjected to differential cell counts (granulocytes/lymphocytes) by means of flow cytometry. Leukocyte sub-populations were analysed for cell viability and changes in cell morphology. The respiratory burst activity was measured with a luminescence assay. Exposure of HKL to SSCM induced an up-regulation of respiratory burst activity after already 1 h, which was still elevated at 24 h, but which was in some cases significantly down-regulated after 96 h. Respiratory burst was positively correlated with the number of live granulocytes in the culture, suggesting that the respiratory burst activity was changed by SSCM effects on granulocyte viability. After 1 h and 24 h of HKL culture, no lymphocyte responses to SSCM were detectable, but after 96 h lymphocyte viability was significantly decreased with SSCM from Spanish S. solidus. In these cultures, residual lymphocytes increased in size, suggesting that cell death and activation might have occurred in parallel. The highest respiratory burst activity was induced by SSCM from Spanish parasites, in particular when they were grown in sympatric sticklebacks. The in vitro HKL responses to SSCM depended on the individual parasite and its population of origin, suggesting that in vivo, S. solidus excretory products are regulated individually, possibly to balance the interplay of each individual host–parasite pair.

Indirubin-3'-Monoxime Induces Apoptosis and Autophagy In Acute Lymphoblastic Leukemia Cells and Chronic Myelogenous Leukemia Cells, Exhibits Limited Cytotoxicity In CD34+ Hematopoietic Stem Cells, Lymphocytes and Granulocytes

AbstractAbstract 3984The rates of treatment-related mortality (TRM) and relapse are unacceptably high in adults undergoing antileukemia treatments for acute lymphoblastic leukemia (ALL). So far has no better therapy with low side effects to improve long-term survival in these patients. Indirubin, a Chinese translational anti-chronic myelogenous leukemia (CML) agent, is able to induce cellular apoptosis. However, until now the functional action of IO on ALL remains still unknown. Therefore, here we investigated and compared the cytotoxic efficacy and action of indirubin-3'-monoxime (IO) in JM1 (ALL cell) and K562 (CML cell). ALL and CML cells were treated with a series of IO dose for 24 and 48h, and cell survival was determined by WST-1 assay. ALL and CML cells were shown to be similar susceptible to IO cytotoxicity. In order to clear in which way of cell death induced by IO, we performed analyses for apoptosis, necrosis and autophagy, respectively. After IO treatment, both ALL and CML cells were arrested in the G2/M cell cycle phase. In addition, an increase of sub-G1 proportion was caused. We found also increasing of caspase-3 activation and formation of cleaved PARP in a dose dependent manner. These were associated with the form of apoptosis. However, the caspase inhibitor Z-VAD-FMK only could partially prevent cell death in ALL and CML cells. When further analyzing the necrotic phenomenon through measuring LDH release, the result clearly showed that LDH release was not remarkable after cell treatment with high dose of IO. Besides, we observed surprisingly in Western blot the increasing expression of microtubule-associated protein light chain 3-II (LC3-II), which generally correlates to formation of autophagosomes. Because better antileukemic drug should not induce toxicity in normal blood cells as much as possible, so the cytotoxic effect of IO in CD34+ hematopoietic stem cells, lymphocytes and granulocytes was analyzed. Excitingly, results showed that IO could not affect cell viability of granulocytes, and IO cytotoxicity in lymphocytes was only marginal. If CD34+ hematopoietic stem cells were treated with IO, the rate of cell survival and their ability of differentiation were almost identical in contrast with non-treated control. Apparently, these data indicated that IO possesses the capability to induce apoptosis and autophagy in both CML and ALL cells. The most important is that the IO hardly influences the cell survival and the differentiation of CD34+ hematopoietic stem cells, the cytotoxic effect in granulocytes and lymphocytes is only limited. In conclusion, IO can be considered as a potential agent for clinical anti- ALL treatment. Disclosures:No relevant conflicts of interest to declare.

Extended storage of granulocyte concentrates mobilized by G‐CSF with/without dexamethasone and collected by bag separation method

The aim of this study was to examine the extended storage of granulocyte concentrates mobilized by granulocyte-colony-stimulating factor (G-CSF) with/without dexamethasone (DEX) and collected by a bag separation method. Ten healthy adult volunteers donated blood three times: twice after granulocyte mobilization by (1) injecting G-CSF at 3 microg kg(-1) subcutaneously (s.c.) and (2) injecting G-CSF at 3 microg kg(-1) s.c. + DEX at 8 mg per oral and once (3) for a baseline control without any forms of mobilization. Granulocytes were collected by a bag separation method. The functions (phagocytosis and oxidative killing levels), viability and levels of interleukin (IL)-1beta, IL-8, IL-6 and tumour necrosis factor-alpha of granulocytes were measured. The average numbers of granulocytes collected from 200-mL samples of whole blood from the G-CSF and G-CSF + DEX groups were 35.1 x 10(8) and 49.4 x 10(8), respectively. Phagocytosis level, oxidative killing level and the viability of the granulocytes mobilized by G-CSF with/without DEX were well maintained for up to 72 h of storage after collection. The levels of the cytokines increased in a time-dependent manner. The in vitro phagocytosis level, oxidative killing level and the viability of granulocytes mobilized by G-CSF with/without DEX and collected by bag separation method can be maintained for as long as 72 h after collection.

The haemoflagellate Trypanoplasma borreli induces the production of nitric oxide, which is associated with modulation of carp (Cyprinus carpio L.) leucocyte functions

In an attempt to characterise the role of nitric oxide (NO) in immune responses of carp, carp leucocytes obtained during an acute T. borreli infection were examined, for their capacity to generate NO. In a second set of experiments the impact NO on viability of the parasite and on the modulation of functional carp leucocyte responses were testedin vitro . Both in carp head-kidneys and in the peripheral blood, the fractions of lymphoblasts among separated leucocytes were increased. However, the relative proportions of granulocytes among head-kidney leucocytes (HKL) significantly decreased during infection, whereas granulocytes appeared among peripheral blood leucocytes (PBL). The cellular dynamics of HKL and PBL of infected carp were paralleled by an enhanced spontaneous NO release in vitro. NO production was further increased after addition of viable parasites to these cultures. The hypothesis that NO had a possible role in granulocyte activation and lymphocyte proliferation in carp was supported by the reduction of mitogen-induced proliferative responses of PBL from healthy carp in the presence of NO donor substances. The negative effects of NO on lymphocyte proliferation were contrasted by enhancing effects on granulocyte functions: the inhibition of NO generation in T. borreli -stimulated HKL cultures by the l-arginine analogue L-NMMA reduced the viability of granulocytes and their phagocytic activity. Even massive amounts of nitric oxide produced by donor substances (up to 600μmol l−1 NO−2) caused no reduction in the numbers of viable T. borreli flagellates in vitro. Thus, in carp, T. borreli seems to induce high amounts of NO in vivo which are apparently not harmful for the parasite but which may interfere with co-ordinated interactions of activated cells aiming at the defence of the parasite.

Effect of metamizol on promyelocytic and terminally differentiated granulocytic cells: Comparative analysis with acetylsalicylic acid and diclofenac

Metamizol is an analgesic and antipyretic agent that can induce agranulocytosis in certain patients. However, its effects on granulocyte viability and differentiation have been poorly evaluated. Here we analysed the effects of metamizol and its active metabolite, 4-methylaminoantipyrine (MAA), on the viability of HL60 promyelocytes and their dimethyl sulphoxide-induced differentiated granulocytes. Metamizol and MAA at 75 μM (above the peak of plasmatic concentration after 2 g intake) did not alter granulocytic differentiation of HL60 cells. Only at concentrations above 100 μM, well over the pharmacological range, metamizol-induced apoptosis in about 30% of the HL60 promyelocytes, while HL60-granulocytic terminally differentiated cells were more resistant to this apoptotic action. When the effects of metamizol were compared with those of acetylsalicylic acid (ASA) and diclofenac on cell viability, at equivalent concentrations used in analgesic and antipyretic therapy (75 μM for metamizol, and ASA and 3 μM for diclofenac) their apoptotic effects were similar. Again, the HL60 promyelocytes were more sensitive to apoptosis than granulocytic differentiated cells, as measured by the percentage of sub-G 1 cells detected by flow cytometry and by determination of caspase activity as a function of poly(ADP-ribose) polymerase cleavage. Furthermore, when human blood-derived granulocytes were treated with metamizol, MAA, and ASA at 75 μM or diclofenac at 3 μM, less than 10% of apoptotic granulocytes were detected, whereas at toxicological/suprapharmacological concentrations (10 mM), about 90% of granulocytes were apoptotic. These results demonstrate that metamizol, MAA, ASA, and diclofenac, at pharmacological concentrations, neither affect the granulocytic differentiation process nor induce relevant apoptosis on terminally differentiated granulocytes.

Optimal storage conditions for preserving granulocyte viability as monitored by Annexin V binding in whole blood

It is important in the laboratory to develop techniques to preserve leucocyte viability in blood specimens for subsequent flow cytometric analysis. This article describes a new simple whole blood lysis method using Annexin V FITC staining which can be used to define both early and late apoptosis of granulocytes in heterogeneous cell populations, without the need for additional stains or to purify the cells (which may result in loss of the cells of interest). The differential Annexin V binding assay was in good agreement with the light microscopy reference method and showed excellent correlation with 7-aminoactinomycin D (7-AAD) staining. It was not affected by problems of morphological interpretation and artifactal changes of granulocyte deformability noted using light microscopy, or the technical difficulties encountered due to red cell contamination using the 7-AAD method. Using this new differential Annexin V staining method, we determined the optimum conditions that maintain granulocyte viability for subsequent flow cytometric analysis and are now employed in our laboratory. These conditions were lithium heparin (Hep) anticoagulated whole blood specimens kept at 4°C with the addition of nutrient medium. Specimens that are anticoagulated with acid citrate dextrose (ACD) or ethyl-diacetyl-tetraacetic acid (EDTA) should also be treated similarly to preserve granulocyte viability and to overcome problems associated with identification of cell populations by flow cytometry.

Heparin in Clinical Doses ‘Primes’ Granulocytes to Subsequent Activation as Measured by Myeloperoxidase Release

The author pre-incubated isolated human granulocytes with increasing doses of heparin (0-100 IU/mL) before subsequent stimulation with formyl-met-leu-phe. There was a dose-dependent increase in granulocyte activation as measured by myeloperoxidase release, quantitated in enzyme-immunoassay. This 'priming' effect of heparin was not due to activation of contaminating platelets or to endotoxin, and there was no reduction in granulocyte viability. Heparin-induced 'priming' of granulocytes may be clinically relevant in the setting of cardiopulmonary bypass. The author describes a sensitive and specific double antibody enzyme-immunoassay for myeloperoxidase from human granulocytes, with an inter-assay coefficient of variation of 12.6%, an intra-assay coefficient of variation of 4.3%, and a lower detection limit of 1.8 microg/l.

89 ASSESSMENT OF CRANULOCYTE VIABILITY AND NBT REDUCTION TEST RESULTS IN CHILDREN LIVING IN AN AREA POLLUTED BY HEAVY METALS

Granulocyte Viability /G.V.T/ and the Nitro-blue-Tetrazolium reduction test /NBT RT/were evaluated in 258 children living in the area of Zinc Smelting Works, Miasteczko Śląskie/Katowice District/ polluted very strongly by heavy metals /Pb, Zn, Cd/. G.V.T. was determined by Cocchi and al. and NBT RT by Raman and al. method. An increase in the percentage of dead granulocytes and a decrease in the value of NBT RT were found, particularly in the youngest children. A large number of examined children showed an elevated Pb and decreased Zn and Mg levels in blood. A correlation was found between Mg and granulocyte viability /t=-3,011, p<0,05/, as well as between Zn and NBT RT values / t = 4,434, p<0,05/. The results of investigations confirm the negative influence of heavy metals polluted environment on intracellular metabolic processes in granulocytes. This phenomenon is highly intensified in the youngest children. The unfavourable Pb influence on children's immunity may be also caused by the interaction of Pb with Mg and Zn in the process of absorption in the intestinal tract.

Human granulocyte antigens: current status and biological significance.

SummaryThe biological reason for the polymorphism of granulocyte-specific antigens is unknown. Clinical complications due to granulocyte antigen-antibody reactions cannot yet be predicted with certainty. Thus reactions following granulocyte transfusions may occur, despite cross-matching (25, 59- 66). Autoimmune disorders and maternal/ fetal incompatibility have been traced to known granulocyte antigens, and sera from patients with these disorders have been used to define many of these antigens (73-86).One of the main difficulties in deciphering the system of granulocyte antigens has been the limitations of the various testing procedures used. None of the techniques yet discovered, including indirect immunofluorescence, has been able to detect all the antigens which other tests detect (28-31). This fact suggests that these tests may be inadequate and could explain their lack of predictive value. A panel of known cell types is also unavailable, because the viability of granulocytes following current cryogen...

Effect of microaggregate blood filtration on granulocyte concentrates in vitro.

To determine the effect of transfusing granulocyte concentrates through microaggregate blood filters, granulocytes prepared with a cell processor were passed through screen and depth microaggregate filters. Pre- and postfiltration evaluations were made of total granulocyte count, levels of muramidase, granulocyte viability, motility, phagocytosis, bactericidal activity, and hydrogen peroxide-forming capacity. Compared to prefiltration levels, a significant (p less than 0.05) decrease in postfiltration granulocyte counts was seen for all the depth filters studied but not for the standard 170 microns (control) or the 40 microns screen filter. For the various tests of granulocyte function evaluated prefiltration, no significant postfiltration differences (p greater than 0.05) were seen for any of the filters studied. Screen microaggregate filters retained only 1 to 3 percent of granulocytes contained in the concentrates, and thus appear satisfactory for use in clinical transfusions. The large percentage of neutrophils retained by the depth filters (20-62%), however, precludes their use for transfusion of granulocyte concentrates.

Stimulatory effect of latex and zymosan particles on cyclic adenosine 3′,5′-monophosphate content in human granulocytes

Human polymorphonuclear leukocytes respond to latex beads and opsonized zymosan particles with increased cyclic AMP formation. The enhancement of cyclic AMP content in cells is related to particle concentration in the incubation medium and to the time of exposure. The temporary stimulation declines after 15 min of phagocytosis, that is in good relation to the uptake of latex particles which reaches saturation point 10 min after the addition of the beads. The cyclic AMP content in granulocytes decreases after between 15 and 30 min of incubation with latex particles. The decrease is not caused by the loss of the granulocyte viability for: (a) the incorporation of labeled methyldeoxythymidine and uridine into macromolecules continues, (b) the sensitivity of phagocytosing granulocytes to prostaglandin E 1 stimulation of cyclic AMP production persists, (c) less than 10% of cells absorbs trypan blue dye after phagocytosis of latex beads. The prostaglandin synthesis inhibitors, aspirin and indomethacin, have no effect on cyclic AMP content in phagocytosing granulocytes.

A test of granulocyte membrane integrity and phagocytic function

An assay of granulocyte viability has been developed which yields information about two important cell parameters, cell membrane integrity and phagocytic activity. The assay is based on the fact that only live cells can accumulate fluorescein, which is enzymatically split from the nonfluorescent substrate fluorescein diacetate. Dead cells, on the other hand, become permeable to the fluorescent red dye ethidium bromide. When cells are exposed first to opsonized zymosan particles, which they can phagocytize, then to a combination of these fluorescent dyes, one can distinguish microscopically between dead cells with fluorescent red nuclei, live cells which fluoresce green and live cells with phagocytic function which are swollen with the pink zymosan particles in a green fluorescing cytoplasm. This assay takes 20–30 min and can be used to distinguish different degrees of cellular damage after cryopreservation.

Viability of granulocytes obtained by filtration leukapheresis.

Nylon filtration alters the morphology of human granulocytes and slightly reduces their phagocytic ability as measured by activation of the hexose monophosphate shunt (HMS) pathway of glucose metabolism. In addition, the ability of rat granulocytes to circulate in granulocytopenic rats is reduced, and this is not attributed to HMS activation during filtration. The activation of the hexose monophosphate shunt is related to the size of the particles rather than to the nature of the material.

The effect of hydroxyethyl starch on in vitro platelet and granulocyte function.

The effect of hydroxyethyl starch (HES) on granulocyte and platelet functions was assessed. No alteration of granulocyte viability, morphology, phagocytic ability, or bactericidal capacity was detected after incubation with 6 per cent HES at 25 C for two hours. Platelet morphology, size distribution, aggregation, nucleotide and serotonin release, and platelet factor-3 availability were also unchanged after exposure to HES. It is concluded that HES has no adverse effect on cell function and appears to be a suitable adjuvant agent for blood cell component collection.

Cryopreservation of human granulocytes

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to −80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol. Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.

Polymorphonuclear Leukocytes Prepared by Continuous-Flow Filtration Leukapheresis: Viability and Function

Abstract Preparation of granulocytes for transfusion in high yield and relatively free of contamination by other cell types has been made possible by the technique of continuous-flow filtration leukapheresis (CFFL). Since previous work suggested that granulocytes collected in this manner may have impaired viability and function, a detailed study of the bactericidal, metabolic, and chemotactic properties of such cells was performed and compared to control cells obtained from the same donors prior to CFFL. The granulocyte percentage of the cell suspensions obtained by CFFL averaged 94.5% ± 1.5% compared to 82.5% ± 1.8% for the controls (p &lt; 0.001) with viability of the PMNs determined by trypan blue exclusion being 97.5% ± 0.9% and 98.2% ± 0.5%, respectively. The phogocytic, metabolic (14C-I-glucose oxidation and protein iodination) and chemotactic properties of both cell types were equivalent in suspensions equalized for granulocyte content. These findings indicate that CFFL technique employed does not impair granulocyte viability or function in vitro. Studies of the in vivo survival and function of CFFL granulocytes are necessary to evaluate their efficacy in combating infection in severely leukopenic patients.