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Biomedical subjects

P Patriarca

Publications and source records attributed to P Patriarca.

At least 55 records · Page 3Linked to original sources
PubMed1985

A simple method to obtain pure granule-rich eosinophil fragments (cytosomes) from normal human blood.

This paper describes a simple, rapid and reproducible method to obtain pure granule-rich eosinophil fragments (cytosomes) with a high yield from normal human blood. The method is based on the treatment of whole blood with saponin and subsequent purification of the cytosomes on Percoll gradient. The enzymatic analysis of the cytosomes shows that the content of 3 granular enzymes is of the same order of magnitude already reported by others for intact eosinophils. This finding suggests that the cytosomes can be employed as starting material for studying the content of the granules or for the isolation of the granular components. The advantages offered by this method over those currently used to obtain eosinophil granules are discussed.

Cell Fractionation↗
PubMed1985

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes. Studies with myeloperoxidase-deficient subjects.

A comparative study of the respiratory burst [monitored as superoxide (O2-) production] of normal and myeloperoxidase (MPO) -deficient polymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O2- production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30-40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O2- produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O2- production by both cell types in response to 4-beta-phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deficient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O2- generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O2- than MPO-deficient PMNs in response to PMA, and the difference in O2- production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O2- generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.

Humans↗
PubMed1985

Increased degranulation of human myeloperoxidase-deficient polymorphonuclear leucocytes.

Myeloperoxidase (MPO)-deficient neutrophils (PMN) released considerably more beta-glucuronidase, lysozyme and vitamin B12-binding activities, when exposed to opsonized zymosan (STZ), than the normal counterpart. Release of the soluble enzyme lactate dehydrogenase was not appreciably changed over the incubation time with particles in either cell type. MPO-deficient PMN and normal PMN ingested STZ particles at a similar rate at early times, but thereafter phagocytosis by MPO-deficient PMN was significantly higher than that by normal PMN. The difference in degranulation between the two cell types greatly exceeded the difference in ingestion and was evident already at early phagocytosis times when no difference in phagocytosis was observed; this suggested that the higher degranulation in MPO-deficient PMN was at least in part independent of the increased ingestion. This was confirmed by experiments with the soluble stimulant N-formyl-L-norleucyl-L-leucyl-phenylalanine (FNLLP). MPO-deficient PMN and normal PMN exhibited a comparable respiratory burst when exposed to FNLLP plus cytochalasin B, but the defective cells released more azurophilic and specific granule markers than normal PMN. These results indicate that MPO-deficient PMN degranulate more than normal PMN and suggest a role for MPO in the regulation of degranulation.

Cytoplasmic Granules↗
PubMed1985

Kinetics of assembly and decay of complement components on Escherichia coli O111:B4 preparation of stable intermediates.

The preparation of bacterial intermediates bearing complement components at various steps of the complement sequence was investigated by suspending immunoglobulin M-opsonized Escherichia coli O111:B4 cells in complement-deficient sera at different temperatures and ionic strengths. The optimal conditions for the formation of the intermediates at Tmax were found to be an ionic strength of 0.091 mu and a temperature of 37 degrees C, except for BAC142, which could be formed equally well at room temperature. In contrast to all the other intermediates, which, once formed at Tmax, were stable in the presence of the whole serum, BAC142 decayed with a half-life of 10 min due to the lability of bound C2. Washing with a buffer of either 0.091 or 0.046 mu did not affect the bacterial intermediates, with the exception of BAC1-3 formed either in the presence of C5-deficient serum or with purified C3 added to BAC142. All the intermediates were found to be stable after incubation in 0.091-mu buffer for 30 min at 37 degrees C.

Complement C3↗
PubMed1984

A simple reliable assay for myeloperoxidase activity in mixed neutrophil-eosinophil cell suspensions: application to detection of myeloperoxidase deficiency.

Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions. Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspension because of the differential inhibition of EPO and MPO by AMT. Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils. The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency. The genealogy derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT. We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

Amitrole↗
PubMed1983

A new method for assessment of serum-induced damage to E. coli.

A simple and rapid spectrophotometric assay for the kinetic evaluation of serum-induced damage to E. coli is described, based on changes in the optical density (OD) of a bacterial suspension. Exposure of antibody-coated E. coli to human absorbed serum results in a diphasic response, namely an increase in OD, which reaches a maximum at about 17 min and is followed by a progressive decrease in OD until a minimum value is reached after 45 min. The increase and the decrease in OD are related to bacterial death and bacterial lysis, respectively.

Blood Bactericidal Activity↗
PubMed1982

Incidence of myeloperoxidase deficiency in an area of northern Italy: histochemical, biochemical and functional studies.

Forty-five subjects with a complete deficiency of myeloperoxidase were identified in an area of the region Friuli-Venezia Giulia in north-eastern Italy using the Hemalog D system as the screening technique. Histochemical and biochemical tests performed on the leucocytes of some of these subjects confirmed the defects shown by the Hemalog D system. The defect was of genetic origin in seven subjects. The genetic origin could be suspected in another eight subjects since more than two affected members were present in a given family. Eosinophil peroxidase, which is present in MPO deficient subjects, interfered with the guaiacol assay of MPO, and in several cases masked the genetic transmission. An assay was developed using o-dianisidine as the electron donor which considerably reduced the interference by EPO. With this assay an autosomal recessive pattern of inheritance was found. The MPO deficient leucocytes had a higher respiratory burst than control cells and an impaired bactericidal activity, at early post-phagocytic periods, which became comparable to that of control cells at later stages. Particle ingestion by the MPO-deficient cells was normal.

Blood Bactericidal Activity↗
PubMed1982

New approaches to the detection of myeloperoxidase deficiency.

Family studies on myeloperoxidase (MPO) deficiency have been carried out by quantitating the peroxidase activity of granulocyte preparations with three methods, namely guaiacol peroxidation, alanine decarboxylation, and spectroscopic analysis. The guaiacol assay failed to show a definite pattern of inheritance in two families with MPO-deficient subjects. Surprisingly, the granulocytes of three histochemically MPO-negative subjects had a peroxidase activity either half or even higher than that of control subjects. The peroxidase activity of these granulocyte preparations in these three subjects showed a positive correlation to the number of eosinophils. The possibility then considered was that eosinophils may have obscured the true pattern of inheritance in this assay. Two other methods of MPO assay, which are not influenced by the presence of eosinophil peroxidase (EPO), were therefore devised. One is based on the ability of MPO, but not EPO, to catalyze decarboxylation of L-alanine in the presence of Triton X-100, and the other relies on the different spectral properties of the two peroxidases. The results obtained with these two methods (1) were strictly comparable, (2) allowed detection of both totally and partially MPO-deficient subjects, (3) differed profoundly from those obtained with the guaiacol method when eosinophil-containing granulocyte preparations were used, and (4) revealed a pattern of autosomal recessive inheritance in the two families studied. The results of the three methods were comparable when eosinophil-free granulocyte preparations were assayed. It is concluded that failure to show a pattern of inheritance in some instances of primary MPO deficiency, or deviations from the autosomal recessive mode of transmission of this defect, may be attributed to interference by EPO. It is proposed that peroxidase assay methods not subject to EPO interference, such as the two described in this article, may be used, particularly in the detection of heterozygote subjects for MPO deficiency in the presence of high eosinophil counts.

Alanine↗
PubMed1981

A two-fold effect of L-1-tosylamide-2-phenylethyl chloromethyl ketone on the oxidative metabolism of guinea pig phagocytes.

The protease inhibitor, L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK), stimulated the O2- production, H2O2 generation, oxygen consumption, and the hexose monophosphate shunt of guinea pig peritoneal polymorphs. Other protease inhibitors were not able to stimulate the metabolic burst of these cells. Maximum stimulation was obtained at 100 microM concentration of the compound. No stimulation was seen in human blood polymorphs even at concentrations higher than those effective on guinea pig polymorphs. TPCK also stimulated the oxidative metabolism of guinea pig blood polymorphs and of guinea pig resident peritoneal macrophages. At concentrations which did not stimulate the oxidative metabolism of guinea pig polymorphs, TPCK inhibited the O2- production induced in these cells by treatment with phorbol myristate acetate (PMA) or with other soluble stimuli. Other protease inhibitors also inhibited the respiratory burst induced by PMA. It is concluded that TPCK exerts two effects on the metabolism of guinea pig phagocytes, which are probably mediated by different mechanisms. The inhibitory effect on the PMA-stimulated respiratory burst might be related to the antiprotease activity of TPCK, while the stimulation of the respiratory burst seems to be independent of protease inhibition.

Amino Acid Chloromethyl Ketones↗
PubMed1981

Evidence that eosinophils catalyze the bromide-dependent decarboxylation of amino acids.

Human eosinophils from subjects with or without myeloperoxidase (MPO) deficiency and guinea pig eosinophils are able to decarboxylate L-alanine in the presence of the cationic detergent cetyltrimethylammonium bromide (CTAB) but not in the presence of the nonionic detergent Triton X-100. Instead, both normal human neutrophils and guinea pig neutrophils decarboxylate L-alanine in the presence of either detergent. When the non-bromide-containing cationic detergent cetyltrimethylammonium hydroxide (CTAOH) is used instead of CTAB, the eosinophils from MPO-deficient subjects are unable to decarboxylate L-alanine. Decarboxylation occurs with the combination CTAOH-Br-, but not with the combinations CTAOH-I-, CTAOH-CI-, or CTAOH-F-. Bromide in the absence of CTAOH does not promote decarboxylation. Triton X-100 and deoxycholate are much less effective in promoting decarboxylation in the presence of bromide. L-Lysine and L-aspartic acid are decarboxylated to a considerably lower rate than L-alanine in the presence of CTAOH and Br-. It is concluded that the eosinophils can catalyze the bromide-dependent decarboxylation of the apolar amino acid L-alanine in the presence of a cationic detergent.

Alanine↗
PubMed1981

Moculating effect of the late-acting components of the complement system on the bactericidal activity of human polymorphonuclear leukocytes on E. coli 0111:B4.

The effect of complement (C) components on the intracellular killing of E. coli 0111:B4 by human PMN was studied. Various intermediate bacteria were prepared by opsonizing IgM-coated 0111:B4 with yeast cell-treated human serum (BAC1-3), a C6-deficient human serum (BAC1-5), a C8-deficient human serum (BAC1-7), a C8-deficient human serum, and with partially purified C8 (BAC1-8). All these bacterial preparations were phagocytosed by human PMN, but only BAC1-8 and, to a lesser extent, BAC1-5 were killed. Similar results were obtained when the 400 x G postnuclear supernatant (PNS) of PMN homogenate was used instead of intact leukocytes. The C9 nature of the killing factor in the PMN homogenate was ruled out by its inability to lyse EAC1-8 and by the finding that the killing of BAC1-8 by the PMN factor was not inhibited by the antiserum against human C9. The anti-C5 and anti-C8 antisera were unable to inhibit the killing by the PNS of BAC1-5 and BAC1-8, respectively, suggesting that bound C5 and C8 do not provide a binding site for the killing factor.

Animals↗
PubMed1979

Effect of antibodies and complement on the interaction between Escherichia coli 0111:B4 and polymorphonuclear leukocytes.

The interaction of Escherichia coli 0111:B4 with polymorphonuclear leukocytes in the presence of specific antibodies and complement was studied. This strain, which is resistant to phagocytosis by polymorphonuclear leukocytes, may be ingested and killed by the phagocytes in the presence of both antibodies and fresh serum. The ineffectiveness of fresh serum to promote ingestion of E. coli 0111:B4 by the phagocytes in the absence of antibodies reflects the inability of this strain to activate the complement system through the alternative pathway. Investigation of the mechanisms of the bacterial killing by polymorphonuclear leukocytes showed that both antibodies and complement were required for the oxygen-independent bactericidal system, whereas they were not needed for the oxygen-dependent system.

Antibodies↗
PubMed1978

Oxidative metabolism of chicken polymorphonuclear leucocytes during phagocytosis.

The oxidative response to phagocytosis by chicken polymorphonuclear leucocytes was investigated as compared to guinea pig polymorphonuclear leucocytes. The polymorphs from both species respond to phagocytosis with an increased oxygen consumption, an increased generation of O2 and H2O2, and an increased oxidation of glucose through the hexose monophosphate shunt. The rate of oxygen consumption, and generation of O2- and H2O2 by phagocytosing chicken polymorphonuclear leucocytes is considerably lower than with phagocytosing guinea pig polymorphonuclear leucocytes. By contrast, the extent of hexose monophosphate shunt stimulation in chicken polymorphs is comparable to that of guinea pig polymorphs. Evidence is presented suggesting that H2O2 is preferentially degraded in chicken cells through the glutathione cycle, whereas catalase and myeloperoxidase are the two main H2O2 degrading enzymes in guinea pig cells. The 20,000 g fraction of the postnuclear supernatant of chicken polymorphs contains a cyanide-insensitive NADPH oxidizing activity which is stimulated during phagocytosis. Similar properties for the NADPH oxidizing activity of guinea pig polymorphs have been previously reported. It is concluded that the metabolic burst of phagocytosing chicken polymorphonuclear leucocytes is qualitatively similar to that of guinea pig polymorphonuclear leucocytes, but the latter cells are more active in all the biochemical parameters that have been measured. The difference in the H2O2 degradation pathways between the two species is accounted for by the lack of myeloperoxidase and catalase in chicken polymorphs.

Animals↗
PubMed1978

[Pathogenicity of E. coli: biological aspects and problems related to laboratory diagnosis (author's transl)].

The biological properties which may contribute to the pathogenicity of E. coli are reviewed in this article. Specifically the following topics are discussed in detail: 1) adhesion to the intestinal epithelial cells, 2) production of enterotoxins, 3) invasiveness of and ability to multiply within the epithelial cells, 4) insensitivity to complement lysis or inability to activate the alternative pathway of complement, 5) resistance to phagocytic killing. The various techniques which might be of potential usefulness in the clinical laboratory to test the parameters of the pathogenicity of E. coli are briefly outlined. The dissociation between the definition of pathogenicity, as established on the basis of the results of E. coli serotyping, and the criteria of pathogenicity listed above is brought to the attention of the reader. As specificity regards the toxinogenicity, what emerged from a survey of the literature, was that only one of the so called "enteropathogenic" strains of E. coli, according to the serotype classification, was found to produce an enterotoxin.

Clinical Laboratory Techniques↗