Antonia Notario

Peptide opioids and morphine effects on inflammatory process

Morphine was found to inhibit human granulocyte aggregation and ATP, thromboxane B2 (TxB2), and leukotriene B4 (LTB4) secretion during cell aggregation. None of the opioid peptides tested [(d-Ala2,d-Leu5)-enkephalin (DADL), (d-Ala2, N-Me-Phe4, Gly-ol5)-enkephalin (DAGO) ordynorphin 1-9 (Dyn 1-9)] was capable of mimicking morphine effects, while Dyn 1-9 per se induced TxB2 and LTB4 secretion from granulocytes. Morphine inhibition of both cell aggregation and ATP, but not of arachidonic acid metabolism product secretion, was prevented by naloxone. The naloxone-sensitive impairment by morphine of CD11b-CD18 complex surface expression observed could play a role in opioid inhibition of granulocyte activation.

The role of integrins in granulocyte dysfunction in myelodysplastic syndrome

The aim of the present study was to evaluate the function of granulocytes in 20 patients affected by myelodysplastic syndrome (MDS) and correlate this with the expression of surface membrane integrins. The granulocytes showed a deficit in chemotaxis (34 +/- 12 vs 84 +/- 10, p < 0.01) in superoxide release (12 +/- 7 vs 30 +/- 10, p < 0.01) and in aggregation 12 +/- 6 vs 36 +/- 9, p < 0.01 using fMLP as stimulus. We also demonstrated with cytofluorimetric and alkaline phosphatase immunoenzymatic analysis (APAAP), decreased expression of CD11b/CD18 receptor detected by OKM1 (p < 0.001) and CD18 detected by MoAb IOT-18 (p < 0.001). PMNs CD11b/CD18 up-regulation and APAAP image analysis studies showed a lower level of expression of CD11b/CD18 in granulocytes from MDS patients compared to controls (p < 0.001). We concluded that granulocyte dysfunction in MDS may be correlated with modification of leukocyte integrins.

The CD11/CD18 granulocyte adhesion molecules in myelodysplastic syndromes

We have evaluated the function of granulocytes in 14 patients suffering from myelodysplastic syndrome (MDS). We also evaluated the functional and immunochemical activities of five monoclonal antibodies (MoAbs) reactive with the CD11/CD18 leucocyte adhesion molecules of granulocytes. Granulocytes showed a decrease in chemotaxis (P < 0.001) and in aggregation (P < 0.01) using various agents as a stimulus. Cytofluorimetric and immunoenzymatic assays with alkaline phosphatase (APAAP) analysis showed decreased expression of the CD11b/CD18 receptor detected by OKM1 (P < 0.001). Despite LFA‐1 and‐CD11a/CD18 was expressed in normal amounts. The studies of upregulation of granulocytes CD11b/CD18 and image analysis of immunochemical preparation (APAAP) demonstrated decreased expression of CD11b/CD18 in granulocytes from MDS compared to controls (P < 0.001). We conclude that granulocyte dysfunction in MDS may be correlated with decreased expression of surface CD11b/CD18 leucocyte adhesion molecules or their structural modification.

Definition of CD 11a, b, c, and CD 18 Glycoproteins on Chemotactically Deficient Granulocyte Membranes in Patients Affected by Myeloid Disorders

We studied neutrophil chemotaxis and surface membrane glycoproteins in 12 patients suffering from myeloid disorders with abnormal karyotype using in vitro techniques in all 12. Chromosome studies were also carried out virtually simultaneously. We chose to study only patients showing a deficit of chemotaxis (p less than 0.001). Ten of these patients revealed a clonal chromosome abnormality in most of the leukemic cells. The monoclonal antibody technique using MoAb 60.1, 60.3, 60.5, OKM1, LFA-1 and 9E8 demonstrated normal leukocyte membrane glycoproteins CD 11a, b, c and CD 18 and therefore excludes that an abnormality of these is involved in the pathogenesis of altered leukocyte chemotaxis. No correlation between chemotactic deficit and any specific clonal chromosome abnormality was found.

The Cytokine Network: Their Role in Physiopathology and Therapeutic Implications

Lymphocytes, accessory-endothelial-and inflammatory-cells produce a variety of soluble factors termed cytokines. These are regulatory molecules which appear after the immune response at all stages of lymphocyte development. They also have important regulatory influences on other haematopoietic cells. Rapid progress is being made through the use of gene cloned cytokines in understanding the complex interactions of these factors with many different cell types. New factors have been added to the cytokine network, and new functions reported for existing cytokines. The clinical implications in disease and the application of cytokines or their inhibitors in human disorders, cancer, infection and autoimmunity, has met with some success and many trials are currently in progress which should lead to more successful therapeutic strategies.

Granulocytes and Myocardial Ischemia

The clinical syndrome of acute myocardial ischemia includes the processes of acute coronary occlusion, possible reperfusion and inflammation (6,8). Each of these processes has been shown to involve granulocytes which disrupt tissues and the contractile machinery of the myocardium (3). The neutrophils are not normally sequestered in the heart; instead they are activated by processes occurring during ischemia and re-oxygenation that lead to their active accumulation. The accumulation of neutrophils is temporally, regionally, and quantitatively associated with the development of myocardial injury (2,14). Drugs, antibodies, or filters that either prevent neutrophil activation (and consequently accumulation) or deplete the blood of neutrophils diminish the area of damage and improve function after ischemia and reperfusion (13). The purpose of this brief report is to discuss the mechanism by which neutrophils may contribute to ischemia-induced myocardial injury and disfunction. Attention will be placed on the potential role of neutrophil-derived mediators in the injurious process, in particular, the metabolites of arachodonic acid (AA) and the oxygen derived free radicals. Recently, these mediators have assumed primary importance because inhibitors of AA lipoxygenation or free-radical scavengers, independently can reduce infarct size.

Membrane Glycoproteins in Superoxide Release from Neutrophils

Neutrophils are the cells generally considered to be the first line of defence against invading microorganisms. Stimulation of their surface receptors activates a repertoire of killing functions as phagocytosis, superoxide production, exocytosis of acid hydrolases and proteases, chemotaxis, aggregation and generation of arachidonic acid-derived inflammatory mediators. The major microbicidal mechanisms of the human granulocytes(PMN) require the formation of reactive oxygen metabolites such as superoxide anion (02) and hydrogen peroxide (the oxidative burst) and the secretion of granule enzymes such as myeloperoxidase (degranulation). Both of these events can be triggered by various soluble or particulate stimuli which interact with the cell surface and depolarize the cell membranes. The PMN to exposed to the specific murine monoclonal antibodies (MoAbs) exhibit a significant depression in (02) generation in response to fMLP to serum-treated zymosan (STZ). Preliminary data suggested that MoAbs depolarizes the PMN cell membrane and that this may be associated with the alterations in oxidative metabolism caused by same MoAbs2’3. The purpose of this study was to explore the relationship between surface membrane glycoproteins, membrane depolarization in the human granulocytes and the subsequent oxidative burst Several MoAbs were used for to identifie the membrane glycoprotein fundamental in respiratory burst activation.

The Pathophysiology of Phagocytes

No survey of clinically important immunological phenomena would be complete without consideration of the functions of phagocytic cells. They play a pivotal role in the immune response by kiling microbes, by presenting antigens to lymphocytes and by serving as supportive, accessory cells to lymphocytes, at least partly by releasing soluble factors. The phagocytes of the body, professional and non professional, consist of two specialized groups of cells: granulocytes, which can be mobilized rapidly and which reach inflamed sites quickly and in large numbers, and which are highly efficient at dealing with many types of injury and infection but which have no capacity for differentiation and live only a short time; and the mononuclear phagocyte system consisting partly of motile cells which respond initially more slowly than neutrophils but which can differentiate in sites of inflammation into cells which are more efficient in various functions than the cells from which they originated. Many mononuclear phagocytes are fixed cells located in tissues where they act as trays or filters for material circulating through the tissue. Phagocytes, which usually function as the primary defender in infections, have also been implicated as effector cells in several conditions characterized by a destructive inflammatory response.

The influence of thyroid hormones on colony growth of peripheral CFU-GM from normal and leukemic subjects.

In order to clarify the alterations of granuloblastic cells in chronic and acute myeloid leukemia, the colony growth behavior of cultured CFU-GM from the peripheral blood of normal and leukemic subjects was examined in basal conditions and after adding to the medium T3 or T4 and/or thioproline and/or flurbiprofen. These drugs had in previous investigations proved their ability to modify cellular receptors and the uptake of thyroid hormones. The study was carried out in semisolid (double agar layer) and liquid medium, utilizing the techniques described previously. Both thyroid hormones enhanced the colony growth from normal peripheral blood CFU-GM and the response was more evident with T4 than T3. The effect on leukemic CFU-GM (from CML and AML) was less clear, probably due to the presence in leukemic cells of a defect of cellular uptake and to the utilization of T3 and T4. Indeed, on addition to the culture medium of thioproline, which modifies membrane permeability, and of fluorbiprofen, which inhibits PGE synthesis, the colony number and growth from leukemic CFU increased considerably in accordance with the results of our previous studies on these substances showing that they are able to modify cellular receptors for thyroid and several other hormones.

The in Vitro Activity of Two Urinary Polypeptides Respect to G- and GM-CSF and IL3 on the Peripheral CFU of Normal and Leukemic Subjects

We isolated two polypeptides on HPLC from the acetonic precipitate of the urine of normal subjects and of patients with untreated AML, APL and AMML. Before separation the quantity of the total urinary factor from leukemic patients was 50±10 mg/24h and from normal subjects was 10±5 mg. We tested the total polypeptidic extract and the two main fractions obtained on liquid cultures of the peripheral CFU of normal and leukemic subjects (AML, APL, CML and CMmL). Colony growth, cell morphology changes and the main cellular markers were examined at the beginning and at the 5th and 10th days of incubation in RPMI 1640 medium. Testing conditions were basal and as determined by the addition of the polypeptidic fractions, both alone and in association with trans-retinoic acid (t-RA) or thioproline (T). Simultaneously and under the same experimental conditions, we tested the activity of G- and GM-CSF and IL3. The results obtained prove the colony stimulating activity of the two fractions and of crude extract; they also prove the ability of such agents to modify the behavior in vitro of peripheral CFU. The urinary extracts are also able to stimulate a moderate differentiation of the elements and an increase both in fibroblasts and in adhesion molecules. We conclude that the dosage of growth factors in urine may be usefull as important marker in several hemopoietic pathological conditions or as a possible source of growth factors.