Biomedical subjects

P Patriarca

Publications and source records attributed to P Patriarca.

At least 19 recordsLinked to original sources

PubMed2000

Role of intracellular chloride in the reversible activation of neutrophil beta 2 integrins: a lesson from TNF stimulation.

The process of beta(2) integrin activation, which enhances the interaction of these heterodimers with ligands, plays a crucial role in the adherence-dependent neutrophilic polymorphonuclear leukocytes' (PMN) responses to TNF. Our previous observation, showing that a marked decrease of the high basal Cl(-) content (Cl(-)(i)) is an essential step in the TNF-induced activation of PMN, stimulated this study, which investigates the role of alterations of Cl(-)(i) in the activation of beta(2) integrins triggered by TNF. Here we show that TNF enhances the expression of activation-specific neoepitopes of beta(2) integrins, namely, epitope 24, a unique epitope present on all three leukocyte integrin alpha subunits, and epitope CBRM1/5, localized to the I domain on the alpha-chain of Mac-1 (CD11bCD18). Moreover, we demonstrate that the conformational changes underlying the expression of the neoepitopes are dependent on a drop in Cl(-)(i) because 1) inhibition of Cl(-)(i) decrease is invariably accompanied by inhibition of beta(2) integrin activation, 2) Cl(-)(i) decrease induced by means other than agonist stimulation, i.e., by placing PMN in Cl(-)-free buffers, activates beta(2) integrins, and 3) restoration of the original Cl(-)(i) levels is accompanied by deactivation of beta(2) integrins. We also show that Cl(-)(i) decrease is required for TNF-induced cytoplasmic alkalinization, but such a rise in pH(i) does not seem to be relevant for beta(2) integrin activation. The results of our study emphasize the role of Cl(-) as a new PMN "second messenger."

Antiporters↗

PubMed2000

TNF-Induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase.

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.

ADAM Proteins↗

PubMed2000

Expression and characterization of recombinant human eosinophil peroxidase. Impact of the R286H substitution on the biosynthesis and activity of the enzyme.

Hereditary eosinophil peroxidase deficiency is a genetic abnormality characterized by a decrease or absence of peroxidase activity and a reduction of the granule matrix volume. Recently, we identified two mutations associated with eosinophil peroxidase deficiency in a subject and his siblings, i.e. a base insertion causing the appearance of a premature stop codon and a base transition causing the replacement of an Arg at codon 286 with a His (R286H). In this article we report the stable expression of both the recombinant wild-type and the R286H eosinophil peroxidase precursor in the K-562 cell line, and the effects of the R286H substitution on the structure and function of the eosinophil peroxidase precursor. Heme group incorporation into both the recombinant wild-type and the recombinant R286H eosinophil peroxidase precursor was comparable, as was the stability of both proteins. Instead, the recombinant R286H eosinophil peroxidase precursor exhibited marked alterations of the catalytic properties and an increased sensitivity to four peroxidase inhibitors with respect to both the recombinant wild-type eosinophil peroxidase precursor and the native enzyme. In addition, the recombinant wild-type, but not the R286H, eosinophil peroxidase precursor was immunoprecipitated by two anti-(eosinophil peroxidase) mAbs. Altogether, our results suggest a protein misfolding of the R286H eosinophil peroxidase precursor which might account for its altered catalytic properties and the absence of expression of some epitopes.

Amino Acid Substitution↗

PubMed1999

Triggering of chloride ion efflux from human neutrophils as a novel function of leukocyte beta 2 integrins: relationship with spreading and activation of the respiratory burst.

PMN residing on immobilized fibronectin have been shown to respond to TNF with an intense and long lasting Cl- efflux that leads to a marked decrease of the unusually high basal Cl- content of these phagocytes. The finding that this Cl- efflux depends, at least in part, on beta2 integrin engagement stimulated the present investigation, which addresses the question as to whether beta2 integrins per se, in the absence of PMN agonists, are able to generate signals triggering Cl- efflux. We induced beta2 integrin cross-linking by plating PMN onto surface-bound mAbs directed against either the common beta-chain (CD18) or the individual alpha-chains (CD11a, CD11b, CD11c) of LFA-1, CR3, and gp150/95. Anti-CD18 mAbs triggered a marked release of Cl- ions, which was accompanied by spreading and activation of the respiratory burst. Cross-linking of gp150/95 and LFA-1 generated the most powerful signals for the activation of Cl- efflux. The results of three independent experimental approaches, i.e., kinetic studies, use of Cl- transport inhibitors, and modulation of Cl- efflux with different amounts of anti-beta2 integrin mAbs, indicated that Cl- efflux regulates both spreading and respiratory burst triggered by beta2 integrin cross-linking. Cl- efflux appears to be independent on either alterations of [Ca2+]i or changes in the plasma membrane potential and shows sensitivity to a raise in pHi. This study uncovers a new signaling ability of beta2 integrins and contributes to highlight the role of Cl- efflux in the outside-in signal transduction pathway regulating adherence-dependent PMN responses.

Antibodies, Monoclonal↗

PubMed1999

Role of the 75-kDa TNF receptor in TNF-induced activation of neutrophil respiratory burst.

The exclusive role of the 55-kDa TNF receptor (TNF-R55) as the signaling receptor in TNF-induced activation of respiratory burst by human polymorphonuclear leukocytes residing on biologic surfaces has been inferred from results obtained with receptor-specific monoclonal and polyclonal Abs. In this work, we confirm this assumption by a more direct approach, i.e., by using receptor-specific TNF mutants (p55TNF and p75TNF) and, as a novel contribution, we show that cooperation of the 75-kDa TNF receptor (TNF-R75) is required for a full blown response to the cytokine. This conclusion stems from three sets of data: 1) none of the TNF-R55-specific agonists used, i.e., mAbs or p55TNF, induced a respiratory burst comparable with that induced by TNF; 2) selective down-modulation of TNF-R75 resulted in a diminished response to TNF but not to TNF-R55-specific agonists or to the chemotactic peptide FMLP; and 3) mAbs that either block or stabilize binding of TNF to TNF-R75 inhibited the response to the cytokine, suggesting that cooperation requires not only TNF binding to the receptor but also an appropriate dissociability from it. The inhibitory effect of the Abs increased as the cytokine concentrations decreased, indicating that cooperation by TNF-R75 becomes more relevant at low TNF doses. Such a cooperation does not seem to rely on the activation of a TNF-R75-linked signaling pathway independent of TNF-R55, since the response to p55TNF and p75TNF given in combination was not higher than the response to p55TNF alone. The possible mechanisms of cooperation are discussed.

Antigens, CD↗

PubMed1997

Biochemical and molecular characterization of hereditary myeloperoxidase deficiency.

Hereditary myeloperoxidase (MPO) deficiency is a neutrophil disorder characterized by the lack of peroxidase activity. Cytochemical, biochemical, spectroscopic, immunocytochemical, and genetic studies were carried out on a 5-year-old MPO-deficient subject and on her parents. The father was also MPO-deficient, whereas the mother had 24% of normal MPO activity. Although the typical absorption spectrum of MPO was absent in both the father and daughter, the father's neutrophils, and not those of the daughter, contained material antigenically related to MPO. In the MPO gene of the father, two mutations were found, each located in a different allele: a T --> C transition, causing the nonconservative replacement M251T and a 14-base deletion within exon 9. The M251T substitution occurred in the carboxy-terminal region of the light chain that is included in the heme pocket. The daughter inherited the 14-base deletion from her father. The study of the MPO mRNAs present in liquid cultures of granulocyte precursors surprisingly showed that the same genetic defect, ie, the 14-base deletion, seemed to exhibit different mRNA phenotypes in the father and the daughter. In fact, mRNA derived from the 14-base-deleted allele was not found in the father and an aberrantly spliced MPO mRNA with a 77-base deletion of exon 9, which includes the 14-base deletion and leads to the generation of a premature stop codon, was found in the daughter. The possibility that Delta77 mRNA could derive from other mutations linked to the Delta14 allele was dismissed because no sequence differences were found in the region (exons and exon-intron junctions). Our data indicate that the alteration of the mRNA context caused by the 14-base deletion provide a basis for the 77-base deletion in the mRNA processing. Since the granulocyte precursors from the liquid cultures of the father were more differentiated than those from the daughter, the observed different behavior of the 14-base-deleted allele in the father and daughter may be the result of a differentiation-stage dependent control of altered spliced mRNA, which may be tolerated during the early stages of differentiation but degraded at later stages. In the liquid cultures of the daughter's cells, in addition to the mRNA with the 77-base deletion, a mRNA with the wild type sequence was also found. This mRNA was inherited from the mother, since no mutations were found in her MPO cDNA and MPO gene. The MPO defect might be caused by a regulatory mutation that induces the MPO gene switch off at an early stage of granulocyte differentiation.

Adult↗

PubMed1997

Biologically active polypeptides in the venom of the jellyfish Rhopilema nomadica.

A tropical jellyfish, Rhopilema nomadica (Scyphozoa, Rhizostomeae) has recently invaded the eastern Mediterranean. Its painful stings have been the bane of bathers and fishermen from Egypt to Turkey. This paper reports on the presence of haemolytic activity and alpha-chymotrypsin-like serine protease activity in the venom of the R. nomadica nematocysts. In addition, the presence of phospholipase A2 activity, which has been described previously, is confirmed. Some properties of these activities are defined.

Animals↗

PubMed1996

Chloride ion efflux regulates adherence, spreading, and respiratory burst of neutrophils stimulated by tumor necrosis factor-alpha (TNF) on biologic surfaces.

Chloride ion efflux is an early event occurring after exposure of neutrophilic polymorphonuclear leukocytes (PMN) in suspension to several agonists, including cytokines such as tumor necrosis factor-alpha (TNF) and granulocyte/macrophage-colony stimulating factor (Shimizu, Y., R.H. Daniels, M.A. Elmore, M.J. Finnen, M.E. Hill, and J.M. Lackie. 1993. Biochem. Pharmacol. 9:1743-1751). We have studied TNF-induced Cl- movements in PMN residing on fibronectin (FN) (FN-PMN) and their relationships to adherence, spreading, and activation of the respiratory burst. Occupancy of the TNF-R55 and engagement of beta 2 integrins cosignaled for an early, marked, and prolonged Cl- efflux that was accompanied by a fall in intracellular chloride levels (Cl-i). A possible causal relationship between Cl- efflux, adherence, and respiratory burst was first suggested by kinetic studies, showing that TNF-induced Cl- efflux preceded both the adhesive and metabolic response, and was then confirmed by inhibition of all three responses by pretreating PMN with inhibitors of Cl- efflux, such as ethacrynic acid. Moreover, Cl- efflux induced by means other than TNF treatment, i.e., by using Cl(-)-free media, was followed by increased adherence, spreading, and metabolic activation, thus mimicking TNF effects. These studies provide the first evidence that a drastic decrease of Cl-i in FN-PMN may represent an essential step in the cascade of events leading to activation of proadhesive molecules, reorganization of the cytoskeleton network, and assembly of the O2(-)-forming NADPH oxidase.

Cell Adhesion↗

PubMed1996

Global eradication of poliomyelitis: benefit-cost analysis.

A benefit-cost analysis of the Poliomyelitis Eradication Initiative was undertaken to facilitate national and international decision-making with regard to financial support. The base case examined the net costs and benefits during the period 1986-2040; the model assumed differential costs for oral poliovirus vaccine (OPV) and vaccine delivery in industrialized and developing countries, and ignored all benefits aside from reductions in direct costs for treatment and rehabilitation. The model showed that the "break-even" point at which benefits exceeded costs was the year 2007, with a saving of US$ 13 600 million by the year 2040. Sensitivity analyses revealed only small differences in the break-even point and in the dollars saved, when compared with the base case, even with large variations in the target age group for vaccination, the proportion of case-patients seeking medical attention, and the cost of vaccine delivery. The technical feasibility of global eradication is supported by the availability of an easily administered, inexpensive vaccine (OPV), the epidemiological characteristics of poliomyelitis, and the successful experience in the Americas with elimination of wild poliovirus infection. This model demonstrates that the Poliomyelitis Eradication Initiative is economically justified.

Child, Preschool↗

PubMed1995

Human eosinophil peroxidase enhances tumor necrosis factor and hydrogen peroxide release by human monocyte-derived macrophages.

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.

Animals↗

PubMed1995

Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis.

A haemolytic toxin was purified by ion-exchange chromatography and FPLC gel filtration from the nematocysts of the jellyfish Carybdea marsupialis. Sheep red cells, but not human or rabbit red cells, were susceptible to lysis by the toxin. The toxin is a protein with an apparent molecular mass of about 102-107 kDa, is heat labile, highly unstable in polar media, inactivated by reducing agents, and devoid of phospholipase activity. The experimental data speak in favour of a pore-forming mechanism of toxin action.

Animals↗

PubMed1995

Leptospira icterohemorrhagiae and leptospire peptidolgycans induce endothelial cell adhesiveness for polymorphonuclear leukocytes.

We have examined the effect of the virulent Leptospira interrogans strain Teramo, serotype icterohemorrhagiae, on the adherence of human neutrophilic polymorphonuclear leukocytes (PMN) to cultured human umbilical vein endothelial cells (HEC). Selective pretreatment of HEC with intact or sonicated leptospires caused a dose- and time-dependent increase of HEC-PMN adhesion (13.2% +/- 2.5% adherence to untreated HEC versus 46.3% +/- 5.6% adherence to HEC pretreated for 4 h with 10(8) intact leptospires per ml [mean +/- standard error of six experiments; P < 0.001]). In contrast, selective leptospire pretreatment of PMN or the addition of leptospires during the adherence assay did not alter HEC-PMN adherence. Leptospire induction of endothelial-cell adhesiveness occurred without detectable HEC damage and was prevented by RNA and protein synthesis inhibitors and by monoclonal antibodies to the CD11/CD18 adhesion complex of neutrophils and to the endothelial-leukocyte adhesion molecule 1 (ELAM-1) of endothelial cells. Similar results were obtained with pretreatment of HEC with interleukin-1 or with the lipopolysaccharide (LPS) of the gram-negative bacterium Escherichia coli. The possibility that contamination by the LPS of gram-negative bacteria could be involved in the induction of HEC adhesiveness was ruled out by the observation that the LPS inhibitor polymyxin B, which abolished the proadhesive effect of E. coli LPS, was ineffective in inhibiting leptospire- as well as interleukin-1-induced adherence. Similarly, leptospire LPSs seemed to have no role in the increase of endothelial-cell adhesiveness, since pretreatment of HEC with a leptospire LPS extract (phenol-water method) or with a leptospire total lipid extract failed to induce the proadhesive phenotype for neutrophils. Instead, peptidoglycans extracted from our leptospires actively stimulated the endothelial proadhesive activity for neutrophils (16.5% +/- 2.1% adherence to untreated HEC versus 51.2% +/- 2.9% adherence to HEC pretreated for 4 h with 1 microgram of peptidoglycan per ml; [mean +/- standard error of four experiments; P < 0.001]). This peptidoglycan-induced activity was inhibited by monoclonal antibodies to the CD11/CD18 adhesion complex and to ELAM-1 but not by polymyxin B. We conclude that peptidoglycans from pathogenic leptospires are among the molecules that can directly activate vascular endothelial cells to increase their adhesiveness for neutrophilic granulocytes. These observations may contribute to a better understanding of the mechanisms whereby non-gram-negative bacteria modulate the local and systemic inflammatory reaction.

CD18 Antigens↗

PubMed1994

Hereditary eosinophil peroxidase deficiency: immunochemical and spectroscopic studies and evidence for a compound heterozygosity of the defect.

Hereditary eosinophil peroxidase (EPO; EC 1.11.1.7) deficiency is a rare abnormality of eosinophil granulocytes characterized by decreased or absent peroxidase activity and decreased volume of the granule matrix. The molecular basis of the defect is not known. We report here its molecular characterization in an EPO-deficient subject and his family members. The EPO-deficient eosinophils contained EPO-related material as determined immunochemically using either monoclonal or polyclonal anti-EPO antibodies but had no spectroscopic evidence of EPO. Eosinophil precursors from the EPO-deficient subject contained normally sized EPO mRNA, which was reverse transcribed into the corresponding cDNA clones encompassing the whole gene. Sequencing of these clones disclosed two mutations, a G-->A transition causing a nonconservative replacement of an arginine residue with a histidine and an insertion causing a shift in the reading frame with the appearance of a premature stop codon. The two mutations were located on different chromosomes indicating a compound heterozygosity for the defect. Both the son and the daughter of the proband inherited the G-->A transition, and their eosinophils contained a peroxidase activity intermediate between that of control subjects and the proband, suggesting that the transition is a deficiency-causing mutation. Eosinophil precursors from the EPO-deficient subject were found to actively synthesize an EPO that was apparently normal in terms of cytochemical reaction for peroxidase and immunoreactivity with monoclonal and polyclonal anti-EPO antibodies, but spectroscopically abnormal. The cytochemical reaction for peroxidase tended to decrease or disappear in the eosinophil precursors of the EPO-deficient subject but not of a normal subject as differentiation went on, suggesting that the Arg-->His substitution causes the production of an unstable EPO that undergoes progressive degradation as the cells mature.

Amino Acid Sequence↗

PubMed1994

Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor.

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF-induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF-induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr-9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin-coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.

Animals↗

PubMed1993

Potentiation of human polymorphonuclear leukocytes respiratory burst and phagocytosis by a standardized liver and spleen fraction of peptides.

The effect of Factor AF2 (AF2), a xenogeneic fraction of peptides with a molecular weight of < 10,000 Dalton obtained from livers and spleens of newborn lambs, on the oxygen consumption and the phagocytic activity of human polymorphonuclear leukocytes (PMN) was studied. AF2 increased the oxygen uptake of PMN exposed both to serum-treated zymosan (STZ), a phagocytosable stimulus, and phorbol-myristate-acetate (PMA), a soluble stimulus. The potentiating effect of the drug was dose-dependent and more pronounced when suboptimal amounts of either stimulus were used. The phagocytic activity of PMN, as measured by the rate of mineral oil particles ingestion, was also increased by AF2 in a dose-dependent manner. These results suggest that the drug may influence PMN behaviour in at least two ways: 1. by increasing the rate of phagocytosis, and 2. by potentiating the respiratory burst induced by soluble and particulate stimuli. The results are discussed in relation to the beneficial effects of AF2 in cancer patients under chemotherapy or radiation treatment.

Adult↗

PubMed1993

Effect of a protein-free dialysate from calf blood on human monocyte differentiation in vitro.

Solcoseryl is a protein-free, standardized dialysate/ultrafiltrate derived from calf blood, which has been shown to improve situations of impaired healing in both experimental animals and man. Its activity seems to be multifactorial although the precise cellular and molecular mechanisms contributing to its effect have not been fully elucidated. Since monocyte-derived macrophages play a central role in inflammation and particularly in wound healing and tissue remodelling, the effect of the dialysate on human monocytes cultured in vitro for 10 days in the presence of human serum was studied. The results show that the drug, at concentrations ranging from 0.2 to 2%, increases the cell density of the cultures and the cell protein content, and favours the differentiation of monocytes into macrophages when 1% serum concentration is used in the culture medium. These effects are no more apparent when the serum concentration was raised to 10%. These data suggest that the drug may substitute, at least in part, for serum in monocyte-macrophage cultures. The observed effects give a sound basis for at least a partial explanation of the therapeutic effects of the drug, particularly at sites where the supply of serum-derived factors is limited.

Actihaemyl↗

PubMed1992

Eosinophil peroxidase deficiency: morphological and immunocytochemical studies of the eosinophil-specific granules.

Five eosinophil peroxidase (EPO)-deficient subjects were identified from 131,000 peripheral blood samples examined for routine automated analysis. The EPO-deficient eosinophils of these subjects met the main criteria established for EPO deficiency: absent or strongly decreased reaction for peroxidase, absent or strongly decreased staining with Sudan Black, and an increased ratio of the granule core volume to the total granule volume. In this report we show that this granule alteration is caused mainly by a decrease of its volume, particularly of the matrix, and that two other matrix proteins, eosinophil cationic protein and eosinophil derived neurotoxin, appear to be present in normal amounts in the EPO-deficient granules.

Adult↗

PubMed1992

A simple and rapid method for isolation of eosinophilic granulocytes from human blood.

A simple and rapid method for the purification of morphologically and functionally intact eosinophils from human blood of both normal and eosinophilic subjects is described. The method is based on a single centrifugation of total blood leukocytes suspended in Percoll with specific gravity 1.0853 g/ml, after erythrocyte removal by dextran sedimentation. The peculiarity of this isolation technique is the maintenance of strictly physiological values of pH and osmolality throughout the entire procedure. Moreover, the cells are not subjected to measures aimed at changing the physical properties of either neutrophils or eosinophils. Because of such characteristics, this isolation method could be usefully exploited for comparative studies of normal and eosinophilic normodense eosinophils and of neutrophils and eosinophils from the same noneosinophilic subject.

Cell Separation↗