An internal standard method for the unattended high-performance liquid chromatographic analysis of ascorbic acid in blood components

A paired-ion, reversed-phase, high-performance liquid chromatography procedure using electrochemical detection and internal standard quantitation based on isoascorbic acid (IA) is described for the determination of ascorbic acid (AA) in blood cells and plasma. By correcting for vial-to-vial variations in the AA oxidation rate, use of IA as an internal standard overcomes a major problem associated with AA instability and eliminates the necessity of assaying samples immediately after they are prepared for analysis. The ion-pairing agent, dodecyltriethylammonium phosphate, gives improved AA-IA resolution over agents with shorter carbon chains and also eliminates the interference of an unidentified substance extracted with platelet AA. Five percent metaphosphoric acid extracts of mononuclear leukocytes (MN), polymorphonuclear leukocytes (PMN), platelets, or plasma were mixed with the IA internal standard and diluted with an EDTA-cysteine solution. The samples were placed in a refrigerated autosampler at 4 degrees C prior to chromatography on a 5-microns octadecylsilyl column. AA concentrations (mean +/- SD) in platelets, MN, and PMN from six healthy volunteers were 0.25 +/- 0.05, 15.2 +/- 6.28, and 2.43 +/- 1.63 micrograms/10(8) cells, respectively; the mean plasma AA concentration was 0.97 +/- 0.34 mg/dl. All are in good agreement with published values. Refrigerated sample extracts containing the internal standard can be reassayed up to 3 weeks later with negligible change in calculated AA concentration. Up to 70 samples can be assayed per day with a detection limit (3 X SD) and minimum quantifiable level (less than 5% coefficient of variation) of 0.02 and 0.2 ng/injection, respectively.     

Antibody-dependent cellular cytotoxicity (AAZZ) against leukemia cells--K-cell activity of human leukocytes

The activity of human leukocytes from the peripheral blood (PB) and bone-marrow (BM) to take the function as K-cells in the antibody dependent cellular cytotoxicity (ADCC) was tested in a 51Cr-test against mouse leukaemic cells and ALL cells covered with specific heterologous antibodies. Mononuclear PB-leukocytes and granulocytes of healthy donors and patients with leukemia and lymphoma in remission lysed murine and human leukaemic cells in the presence of specific antibodies. There was no lowering of K-cell activity of mononuclear PB-leukocytes of patients with leukaemia and lymphoma in remission under chemotherapy as compared with healthy donors and patients in remission without chemotherapy. There was a good correlation between the percentage of K-cell active mononuclear leukocytes in PB and BM. Attempts of fractionation with peripheral blood leukocytes of healthy donors resulted in the non-adherent mononuclear PB-leukocytes (lymphocytes) and granulocytes being effector cells in ADCC. To a high degree K-cell active lymphocytes could be identified in the non-B-fraction and only slightly in the fraction forming E rosettes.     

Composition and metabolism of phospholipids of human leukocytes

Human mononuclear (MN) and polymorphonuclear (PMN) leukocytes were analyzed for their phospholipid, triglyceride, cholesterol and fatty acid content. The phospholipid/cholesterol ratio was 1.24 for both cels. MN cells contain more phosphatidylcholine (PC), but less phosphatidylserine (PS), phosphatidylethanolamine (PE) and sphingomyelin (SPH) than PMN cells when expressed as percent of total phospholipid. When expressed on the basis of lipid content per cell, MN cells contain less PS, PE and SPH but more triglyceride than PMN cells. PMN cells incorporate palmitic, stearic, linoleic and linolenic acids into their phospholipids, triglycerides or cholesterol esters. The incorporation into triglycerides was highest for all fatty acids. Of the phospholipids, the incorporation was highest into PC. Labeled fatty acids also were found in proteins which had been delipidized by exhaustive extraction with organic solvents. These represent tightly or covalently bound fatty acids. The incorporation of [³H]palmitic acid into this protein fraction is stimulated by insulin.     

An indirect bioluminescence method for the quantitative measurement of polymorphonuclear cell chemotaxis

A method is presented which allows the quantification of the effects of chemotactic factors on polymorphonuclear leukocytes on the basis of a sensitive ATP measurement using bioluminescence. The assay measures those cells which have migrated through a commercial 3 μm filter system (Transwell?). The assay was tested under standardized conditions with different chemotactic agents (leukotriene B4 [LTB4], N-formyl-methionyl-leucyl-phenylalanine [FMLP], N-formyl-methionyl-leucyl-phenylalanine-methyl ester [M-FMLP]). Under appropriate conditions the migration of PMN-cells is time-dependent and linear for 60 minutes. Spontaneous migration of PMN cells is simultaneously quantified in a simple way, and the value obtained allows a determination of the actual chemotactic stiuation of the PMN cells. In healthy humans the spontaneous migration varied between 4.2% and 14.4% of the total number of PMN cells. An optimal chemotactic activity was detected at 10^?8/mol/I for FMLP and 10^?7 mol/l for M-FMLP in PMN leukocytes, which correlates with literature values. It was also found that in contrast to EDTA blood, heparinized blood lowers the ATP level of PMN cells (by about 50%) and therefore heparinized blood is not recommended for chemotactic experiments. This assay is a simple tool for quantification of the spontaneous migration, and the chemotactic response to specific factors and their inhibitors in particular for pharmacological experiments. In contrast to the ‘classical’ chemotactic assays this method also permits the simultaneous testing of the influence of chemotactic substances on cellular ATP levels.     

Flow cytometric analysis of glyoxalase-1 expression in human leukocytes

Altered glyoxalase‐1 (GLO‐1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress‐ and aging‐related diseases. In the present study, we developed a flow cytometry method for GLO‐1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO‐1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO‐1 in leukocytes showed that expression index of GLO‐1‐positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO‐1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO‐1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress‐related disorders. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of epoxide hydrolase and glutathione S-tranferase genotypes on the induction of micronuclei and DNA damage by styrene-7,8-oxide in vitro

Styrene is one of the most important organic chemicals used worldwide. Its main metabolite, styrene-7,8-oxide (SO), is considered responsible for the genotoxic effects associated with exposure to styrene. SO is detoxified by hydrolysis catalyzed by epoxide hydrolase (EH), or, to a minor extent, by conjugation mediated by glutathione S-transferases (GSTs). The purpose of the present study was to investigate whether EH (exons 3 and 4), GSTP1 (exons 5 and 6), GSTM1 and GSTT1 polymorphisms have any influence on the genotoxicity of SO in human leukocytes. Peripheral leukocytes from 30 healthy donors were exposed to SO (50 and 200 micro M) and genotoxicity was evaluated by means of the micronucleus (MN) test and alkaline comet assay, using 1% DMSO as solvent control. When EH genotypes were classified in low, medium, and high with respect to the expected EH activity, an increase in induced comet tail length was observed with decreasing EH activity in SO-exposed cells. An increase was seen in induced MN frequency in EH low-activity donors. These findings are consistent with the detoxifying activity of this enzyme. In addition, increases in MN frequencies for GSTP1 *A/*B and *A/*C genotypes with regard to the wild-type homozygous *A/*A genotype were detected. This may be due to a low detoxifying activity as a consequence of altered SO affinity of the variant protein, but must be confirmed using homozygote variant individuals, not included in this study. No clear results were obtained for GSTM1 or GSTT1 genotypes, even when performing the analysis after grouping individuals with the same expected EH activity, probably due to the minor role that glutathione conjugation plays in styrene metabolism. The present in vitro findings using human leukocytes suggest that polymorphisms in EH, and, to a lesser extent, in GSTP1, may influence induction of cytogenetic and DNA damage by SO.     

Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.     

Oxygen free radical producing activity of polymorphonuclear leukocytes in patients with Parkinson's disease

Oxygen free radicals (OFRs) have been suggested in the pathogenesis of Parkinson's disease (PD). These free radicals exert their cytotoxic effect by peroxidation of lipid membrane resulting in the formation of malondialdehyde (MDA). Polymorphonuclear (PMN) leukocyte is one of the major sources of OFR. However, the oxygen free radical producing activity of PMN leukocytes in patients with PD is not known. We therefore studied the oxygen free radical producing activity of polymorphonuclear leukocytes and MDA levels in the serum of healthy subjects and in patients with Parkinson's disease. The oxygen free radical producing activity of PMN leukocytes in blood and the MDA content in serum were significantly higher in patients with Parkinson's disease than in healthy subjects. These results indicate a possible role of oxygen free radicals in the pathogenesis of Parkinson's disease.     

Enhanced killing of Pseudomonas aeruginosa by human polymorphonuclear leukocytes in presence of subinhibitory concentrations of carbenicillin and ticarcillin

The effect of carbenicillin and ticarcillin on the killing of Pseudomonas aeruginosa was studied with an in vitro system using peripheral blood polymorphonuclear (PMN) leukocytes collected from human donors. No corticosteroid was given to the donor prior to leukocytes collection by a continuous flow cell separator. The assay was carried out with or without serum. P. aeruginosa yield after a 4 hour-incubation was estimated by colony counting. In Hanks' balanced salt solution, P. aeruginosa strains 74 and 78 were resistant to human PMN leukocytes. The presence of subinhibitory concentrations of carbenicillin or ticarcillin (1/10th the minimal inhibitory concentration (MIC) for P. aeruginosa 74, 1/4th the MIC for P. aeruginosa 78) enhanced the bactericidal activity of human leukocytes. Difference between the numbers of bacteria recovered with PMN cells and without cells increased with concentration of carbenicillin or ticarcillin. The synergistic effect was not observed when serum (heated fetal calf serum or heated pooled human serum) was used. The mode of action of carbenicillin and ticarcillin on bactericidal activity of phagocytic cells was not elucidated, but we suggest the effect is due not to action on the phagocytic cells themselves but on the microorganisms.     

The dual functionality of antimicrobial peptides Os and Os‐C in human leukocytes

Antimicrobial peptides (AMPs), Os and Os‐C, have been identified as multifunctional peptides with antibacterial, antiendotoxin, and anti‐inflammatory properties. For further development of Os and Os‐C as therapeutic peptides, it is essential to evaluate these effects in human mononuclear (MN) and polymorphonuclear (PMN) leukocytes. The cytotoxicity and the effects of both peptides on MN and PMN morphology were determined with the Alamar‐Blue assay and scanning electron microscopy, respectively. The ability of Os and Os‐C to induce reactive oxygen species (ROS) and to protect against 2,2′‐azobis(2‐amidinopropane) dihydrochloride–induced oxidative damage in both cell populations was evaluated using 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA). Using fluorescently labeled peptides, the ability of the peptides to cross the cell membranes of MN and PMN was also evaluated. At the minimum bactericidal concentrations of Os and Os‐C, neither peptide was cytotoxic. Os caused morphological features of toxicity at 100 μM, entered MN cells, and also protected these cells against oxidative damage. Os‐C caused MN and PMN leukocyte activation associated with ROS formation and was unable to penetrate cell membranes, indicating extracellular membrane interactions. This study confirms that both Os and Os‐C at less than 100 μM are not cytotoxic. The MN‐specific uptake of Os identifies it as a cell‐specific cargo‐carrier peptide, with additional anti‐inflammatory properties. In contrast, the ability of Os‐C to activate MN and PMN cells implies that this peptide should be further evaluated as an AMP, which, in addition to its ability to eradicate infection, can further enhance host immunity. These novel characteristics of Os and Os‐C indicate that these AMPs as peptides can be further developed for specific applications.     

An evaluation of the metabolic basis of aflatoxin B1 toxicity by using buffalo granulocytes and agranulocytes in vitro

This study was aimed at monitoring cytotoxic changes in buffalo leukocyte subpopulations exposed to aflatoxin B1 (AFB1), since no such information is available for this species. The effects of AFB1 on glutathione (GSH) S-transferase, Ca2+Mg2+ATPase and protein synthesis in leukocyte subpopulations, namely, mononuclear (MN) cells and polymorphonuclear (PMN) cells isolated from the blood of the domestic buffalo (Bos bubalis), were studied. The cells were separated by using Ficoll-Paque and incubated in the presence of AFB1. GSH S-transferase activity was found to increase in cells exposed to AFB1, but there was no difference in activity between MN and PMN cells. PMN cell ATPase activity increased after AFB1 treatment, whereas no such effect was observed in the MN cells, which showed higher basal levels of ATPase activity. In the presence of AFB1, all the cells showed significant decreases in 14C-leucine incorporation, but the MN cells showed higher 14C-leucine incorporation than the PMN cells. Nevertheless, both cell types were affected by AFB1 and participated in its detoxification. There was also an appreciable decrease in the release of myeloperoxidase by activated PMN cells in the presence of AFB1.     

Metabolic activity of phagocytes in experimental sporotrichosis

Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to agressive microorganisms. In patients with sporotrichosis a diminished capability of killing engulfed yeast by their PMN has been described, but the origin of this deficiency remains unknown.In this work, partial aspects of the oxidative metabolism of PMN leukocytes and peritoneal macrophages of mongolian gerbils experimentally infected with sporotrichosis were studied. For this purpose the nitroblue tetrazolium (NBT) test as described by Baehner and Nathan (1) and myeloperoxidase activity measured according to Kaplow's method were utilized.The PMN and macrophages of mongolian gerbils infected with sporotrichosis showed increased reduction of NBT when compared with the phagocytic cells of normal ones, as is usually observed in most infections. Myeloperoxidase activity was diminished in both PMN and macrophages, but this diminution was statistically significant only in PMN leukocytes. These results show that part of the oxidative mechanisms of phagocytic cells can be impaired in experimental sporotrichosis, and could be correlated with the diminished fungicidal activity of PMN leukocytes obtained from patients infected with sporotrichosis.     

Activation of the phosphatidylinositol-3'-kinase pathway with lectin-induced signal through sialo-containing glycoproteins of leukocyte membranes under type 1 diabetes mellitus

The influence of wortmannin and sialospecific lectins on the translocation of p85alpha regulatory subunit of phosphatidylinositol-3'-kinase (PI-3'-kinase) between membrane and cytosolic fractions of the mononuclear and polymorphonuclear leukocytes in healthy donors and patients with type 1 diabetes mellitus (DM) was investigated. It was found out that under type 1 DM PI-3'-kinase takes active part in the transduction of lectin-induced signal through membrane glycoprotein receptors that contain terminal sialic acids linked to subterminal carbohydrate residues with (alpha2-->6) glycosidic bond.     

Impaired reactive oxygen metabolism of phagocytic leukocytes in NIDDM patients. A role for non-enzymatic glycosylation of collagen

Non-enzymatic glycosylation (NEG) of collagen has been previously shown to significantly influence the reactive oxygen metabolism (ROM) of phagocytic cells in healthy subjects. Considering the role of NEG in the pathophysiology of diabetes, we have further analysed the oxidative metabolism of polymorphonuclear cells (PMNs) and monocytes in 23 patients with non-insulin dependent diabetes mellitus in order to better elucidate a possible pathogenic role of NEG of the extracellular matrix in long-term complications of diabetes. Experiments were performed in triplicate on native-collagen and glycated-collagen coated vials, using a chemiluminescence (CL) assay. Results show that PMNs from diabetic patients display a significant increased basal and zymosan-induced CL activity with respect to controls that are not related to the glycation state of the substrate. Conversely, the CL activity of monocytes induced by zymosan shows a decrease in diabetic patients with respect to healthy volunteers (p < 0.05). Moreover, monocyte CL was reduced by the glycated matrix, both in healthy volunteers and in diabetic subjects (p < 0.05 and p < 0.01, respectively). These data highlight a complex role of phagocytic leukocytes in the pathophysiology of extracellular matrix alterations secondary to NEG that are typically present in clinical conditions such as diabetes or ageing. © 1998 John Wiley & Sons, Ltd.     

Monitoring of polymorphonuclear leukocyte functions in diabetes mellitus— a comparative study of conventional radiometric function tests and low-light imaging systems

In this study neutrophil (PMN) phagocytic capacity was investigated using a conventional radiometric ingestion assay (IN) in comparison with PMN respiratory burst activity assessed by luminol-enhanced chemiluminescence (LCL) in response to phorbolesters and LCL induction during phagocytosis of opsonized Staphylococous aureus (STLCL) in diabetes mellitus and healthy controls. PMN ingestion was measured with ³H-thymidine-labelled S. aureus in a kinetic radiometric assay. LCL and STLCL were assessed in a parallel detecting microtitre-plate luminometer (MTP-Reader). PMN of diabetic subjects showed a highly significant reduction of peak LCL in response to PMA as well as during phagocytosis of S. aureus (STLCL) compared to non-diabetic controls (p<0.001 respectively). PMN ingestion in diabetic patients (51.8±4.6%) was significantly reduced compared to controls (78.3±6.2%) (p<0.01). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations ? 13.8mmol/L, whereas PMN IN was significantly reduced at glucose levels ?27.75mmol/L. The control group showed a positive correlation of peak LCL response and IN (p<0.05) but not of STCL and IN; in diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN respiratory burst activity and markedly reduced phagocytic PMN functions in diabetic patients ex vivo and in vitro as measured by LCL and by ingestion of ³H-thymidine-labelled S. aureus suggesting inhibitory effects of elevated glucose concentrations on various PMN-functions, which might be of clinical importance concerning altered host defence.     

Apoptosis and oxidative status in peripheral blood mononuclear cells of diabetic patients

We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type I (IDDM) or non insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 and CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione (GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma glutamyltransferase (-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC from diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be used to identify the onset of diabetes.     

Effect of single oral administrations of non steroidal antiinflammatory drugs to healthy volunteers on arachidonic acid metabolism in peripheral polymorphonuclear and mononuclear leukocytes

The effects of a single oral administration of acetylsalicylic acid (500 mg), indomethacin (50 mg) and piroxicam (40 mg) to healthy volunteers on functional and biochemical parameters of platelets, polymorphonuclear (PMN) and mononuclear (MNL) leukocytes were evaluated. Blood was collected before and two hours after the drug intake and blood cells separated according to conventional techniques. The considered drugs almost completely suppressed the aggregation of platelets, whereas they did not affect either PMN and MNL aggregation. Superoxide anion generation by leukocytes was (PMN), or no effect (MNL) was observed after piroxicam and indomethacin respectively. The formation of arachidonate metabolites via the 5-lipoxygenase pathway by PMN and MNL challenged with 10 microM A23187 was unchanged following aspirin and indomethacin. In this respect a selective increase of 5-HETE and LTC4 synthesis by MNL only was detected after piroxicam administration. The three drugs similarly reduced TXB2 synthesis by platelets and PMN (-80% for aspirin and indomethacin, and -40% for piroxicam). As far as MNL is concerned, aspirin inhibited this metabolite by 80%, while indomethacin reduced it by 40% only. In contrast piroxicam increased TXB2 synthesis by stimulated MNL. It can be concluded that the considered antiinflammatory drugs 1) differently affect the cyclooxygenase enzyme in platelets and leukocytes; 2) at variance with the situation in platelets, the inhibition of thromboxane formation by leukocytes is not related to modifications of cellular function; 3) the formation of arachidonate metabolites via the 5-lipoxygenase pathway is affected by piroxicam only.     

Circadian variation of cytosol glucocorticoid receptors in human polymorphonuclear leukocytes (PMN) and mononuclear cells (MN) in a normal population

Glucocorticoid cytosol and whole cell receptors from human PMN's have been quantified, and compared to those of human MN leukocytes on the same blood sample. The normal cytosol PMN receptor density (N = 15) averaged 1,254 +/- 105 (SE) molecules bound/cell at 0900 h and increased significantly to 1,497 +/- 98 at 2,100 h (P less than 0.02). MN cell cytosol receptor density was 1,198 +/- 145 at 0900 h and increased significantly to 1,551 +/- 117 molecules bound/cell at 2,100 h (P less than 0.01). Corresponding whole cell receptor densities at 0900 h were 2,845 +/- 273 (PMN) and 3,547 +/- 290 (MN) and these did not change significantly at 2,100 h. Conclusions: Cytosol receptors in normal human PMN and MN cells increased significantly at 2,100 h from the 0900 h level while serum cortisol levels were dropping. Whole cell receptors in the same PMN and MN cell samples did not change significantly between 0900 and 2,100 h. The normal circadian variation in serum cortisol influences the distribution of the glucocorticoid receptor between the cytosol and the nucleus, but does not influence the amount of receptor available to the whole cell. This is the first time that an endogenous physiological variation of cortisol concentration has been utilized to demonstrate a corresponding change in receptor capacity in vivo.     

Intraleukocytic Bactericidal Activity in Patients Receiving Corticosteroid and Radiation Therapy,and in Patients with Diabetes Mellitus

Bactericidal activities of peripheral white blood cells obtained from patients and from healthy persons were examined in vitro. The results obtained are summarized as follows. 1. Peripheral white blood cells from patients receiving corticosteroid and radiation therapy showed decreased levels of intracellular bactericidal activities against Staphylococcus aureus. The leukocytes from almost all patients examined displayed intense activities of intracellular bacterial killing against Streptococcus pyogenes. 2. Only polymorphonuclear leukocytes (PMNs) and macrophages obtained from patients in severe stages of diabetes mellitus exhibited decreased levels of intracellular bactericidal activities against S. aureus. 3. The leukocytes from all patients examined exhibited the same levels of intracellular bactericidal effects against S. pyogenes as leukocytes from healthy persons. 4. Pseudomonas aeruginosa, which was phagocytized by PMNs obtained from healthy persons, demonstrated a remarkable degree of resistance to any intracellular bactericidal effect.     

Lactoferrin: affinity purification from human milk and polymorphonuclear neutrophils using monoclonal antibody (II 2C) to human lactoferrin, development of an immunoradiometric assay using II 2C, and myelopoietic regulation and receptor-binding characteristics

Several investigators have now confirmed our original report demonstrating the myelopoietic suppressive activity of lactoferrin (LF) in vitro. In order to further clarify this activity, we used the recently produced and purified neutralizing antibody (II 2C) to LF to set up an immunoradiometric assay specific for LF and to affinity purify LF from lysates of peripheral blood polymorphonuclear neutrophils (PMN) obtained from healthy donors. Iron-saturated purified PMN LF was as active as iron-saturated affinity purified milk LF as a suppressor of the release of granulocyte-macrophage colony stimulating factors (GM-CSF) from mononuclear human peripheral blood leukocytes. The activities of both the PMN LF and milk LF were inactivated by preincubation with monoclonal anti-LF antibody (II 2C). In order to evaluate the methods of iron saturation of LF in vitro as measures of their functional activities, milk LF was iron saturated by four different methods, including ferric citrate, ferric ammonium sulphate, ferric chloride with nitriloacetate, and ferric chloride alone. The functional characteristics of all four preparations of LF saturated with iron in vitro were relatively equal and were more active than native LF. Resident mouse peritoneal macrophages separated into subpopulations of GM-CSF-producing cells by velocity sedimentation were evaluated for their LF-receptor binding capacity and for sensitivity to the suppression of GM-CSF release by LF. Iron saturated LF suppressed release of GM-CSF from only those fractions containing LF-receptor bearing cells, although not all fractions containing cells bearing receptors for LF responded to the suppressive activity of LF. These studies provide further evidence for the myelopoietic regulatory activity in vitro of PMN-derived LF, which is mediated through populations of mononuclear phagocytes having receptors for LF.