Reduction of blood coagulation and monocyte-platelet interaction following the use of a minimal extracorporeal circulation system (Synergy) in coronary artery bypass grafting (CABG).

Cardiovascular surgery with cardiopulmonary bypass (CPB) induces activation of blood coagulation and systemic inflammation involved in post-operative complications. Our study evaluated the impact of the minimal extracorporeal circulation (mini-CPB) system (Synergy, Sorin Group) on these functional aspects. Twenty patients were randomly assigned to standard CPB (n = 10) or to Synergy (n = 10). Platelet expression of PAC-1, and monocyte/granulocyte-platelet conjugates were evaluated by flow cytometry. A leukocyte-platelet adhesion index was calculated after cell number normalization. ELISAs were performed to measure IL-6 and TNF-alpha, thrombin-antithrombin III complexes (TAT), prothrombin fragments (F1+2), beta-thromboglobulin (beta-TG) and sP-selectin (sCD62P). Blood samples were drawn at the time of anesthesia (T1), at the end of CPB (T2), and at 4 (T3) and 24 hours (T4) after weaning from CPB. All patients were similar for clinical characteristics. When compared to standard CPB, the Synergy showed lower levels of the monocyte-platelet adhesion index at T2 (0.023 +/- 0.005 vs 0.063 +/- 0.013, P = 0.0092) and T4 (0.031 +/- 0.003 vs 0.055 +/- 0.005, P = 0.0017), TAT complexes at T2 (27.175 +/- 5.967 vs 86.592 +/- 5.415, P = 0.0005) and T3 (26.977 +/- 2.468 vs 45.146 +/- 4.365, P = 0.0041), F1+2 fragments at T2 (2.222 +/- 0.226 vs 4.249 +/- 0.292, P = 0.0009), and sP-selectin at T3 (115.17 +/- 19.623 vs 169.554 +/- 19.709, P = 0.0703) and T4 (108.542 +/- 6.429 vs 140.799 +/- 14.771, P = 0.0833). In summary, the Synergy exhibited a lower post-operative activation of blood coagulation, together with a reduced interaction between circulating monocytes and platelets.

Purpurin-18 in combination with light leads to apoptosis or necrosis in HL60 leukemia cells.

Photodynamic therapy (PDT), a cancer treatment using a photosensitizer and visible light, has been shown to induce apoptosis or necrosis. We report here that Purpurin-18 (Pu18) in combination with light induces rapid apoptotic cell death in the human leukemia cell line (HL60) at low doses and necrosis at higher concentrations. Cells treated with Pu18 and light under apoptotic conditions exhibited DNA laddering and an increase in both cellular content of subdiploid DNA and externalization of phosphatidylserine (PS), indicating DNA fragmentation and loss of membrane phospholipid asymmetry. In the absence of light activation, Pu18 at nanomolar concentrations had no detectable cytotoxic effect. Caspase-3 activity was increased even after 1 h from treatment with low doses of Pu18 and light. The PS exposure and nuclear features of apoptosis were prevented by treatment of cells before illumination with caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK). Conversely, the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) failed to suppress the apoptosis. No protective effect of the three caspase inhibitors was observed when the cells were exposed to necrotic concentrations of Pu18 and light. Our results show that caspase-3, but not caspase-1, is involved in the signaling of apoptotic events in PDT with Pu18-induced apoptosis of HL60 cells. Moreover, both the time course of PS exposure and the effect of caspase inhibitors on it indicate that it is regulated in the same manner as DNA fragmentation.

Detection of eosinophils in whole blood samples by flow cytometry.

A flow cytometric method to detect and study human eosinophils in whole blood was established. Normal subjects and patients with various types of eosinophilia (hypereosinophilic syndromes, allergic diseases, dermatitis, Hodgkin's Disease, parasitosis) were studied. Whole blood samples were treated for 10 minutes at room temperature with a commercially available reagent (FACS Lysing Solution, Becton Dickinson) which acts both as a fixative and as a lysing agent. Eosinophils were identified as a granulocytic subpopulation with higher SSC and FSC properties. This cell population was characterized by evident autofluorescence and hypodiploid DNA features after propidium iodide staining. The purity of the eosinophil population sorted after electronic gating was close to 100%. A very significant correlation between eosinophil counting by our whole blood method and other two assays, namely routine automatic counting by the H*3 Bayer System and eosinophil detection by depolarized SSC, was obtained. The phagocytic properties of eosinophils were also studied by means of a commercially available diagnostic kit, thus demonstrating that our method is also suitable for the study of those granulocytic functions which can be evaluated by flow cytometry.

fMLP-induced CD11b/CD18 upregulation on neutrophils from patients with non-Hodgkin's lymphomas treated with recombinant human granulocyte colony-stimulating factor.

The effects of rhG-CSF administration on fMLP-induced neutrophil CD11b and CD18 upregulation were studied in nine patients suffering from intermediate and high grade non-Hodgkin's lymphomas. Blood samples were obtained before recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration and 24 hrs after rhGSF interruption. The growth factor was administered subcutaneously for five days in a dosage of 5 microg/Kg/day. Nine normal subjects were studied as controls. Five patients showed an impaired baseline CD11b and CD18 upregulation, which was corrected by rhG-CSF therapy. Four patients showed a normal baseline CD11b and CD18 upregulation, but this function was reduced by rhG-CSF therapy. All patients showed a normal baseline fMLP-induced luminol-enhanced chemiluminiscence and significantly increased chemiluminescence values after rhG-CSF administration. We conclude that, while in some patients rhg-CSF is able to improve neutrophil CD11b and CD18 upregulation in response to chemotactic agents, in other patients a decrease of this function can occur, maybe due to a relative immaturity of the circulating neutrophils induced by rhG-CSF.

Actin polymerization in neutrophils from patients affected by myelodysplastic syndromes--a flow cytometric study.

In this study F-actin polymerization in neutrophils from 21 patients affected by myelodysplastic syndromes (MDS) was evaluated by means of a flow cytometric assay. Neutrophils were stimulated with formyl-methionyl-leucyl-phenylanaline (fMLP; 10(-8) M final concentration) for 15, 30, 60 and 120 sec, and F-actin content was determined using fluorescein-isothiocyanate phallacidin as a specific probe. Eight normal subjects were studied as controls. We found that F-actin polymerization was defective in ten patients, with very impaired values after 60 and 120 sec of stimulation with fMLP. The remaining 11 patients showed a prevalent neutrophil population with normal F-actin polymerization and neutrophil sub-populations with either defective or undetectable F-actin polymerization. In the first group, patients with very poor prognosis (refractory anemia with excess blasts, refractory anemia with excess blasts in leukemic transformation, trisomy 8, multiple karyotypic abnormalities) were present, although patients with aberrations of karyotype were present in the second group. It is possible that defects in neutrophil F-actin polymerization may be responsible for neutrophil dysfunction, which has frequently been observed in MDS.

Granulocyte colony-stimulating factor (G-CSF) administration increases PMN CD32 (FcRII) expression and FcR-related functions.

The phenotypical and functional properties of circulating neutrophils from ten patients suffering from intermediate- and high-grade non-Hodgkin lymphoma were investigated before and after rhG-CSF administration (5 micrograms/kg/day subcutaneously for 5 days). The following parameters were studied: flow cytometry evaluation of surface CD32, CD16, CD11b and CD18 by means of a whole blood method; whole blood phagocytosis by means of a flow cytometric assay; whole blood chemiluminescence using opsonized zymosan as a stimulus. A significant increase in the expression of surface CD32 was detected in all patients, while CD11b expression was found to be increased in only four of them. CD16 and CD18 expression did not change. A significant enhancement of phagocytosis and phagocytosis-associated chemiluminescence was also observed. These results show that rhG-CSF administration can increase both FcRII expression and FcR-related functions.

Effects of lysine-arginine association on immune functions in patients with recurrent infections.

Combined L-lysine-L-arginine therapy is capable of inducting recovery in age-related decline of thymic activity in mice and in elderly humans. The clinical usefulness of the association has also been shown in children with recurrent respiratory infections, while an increase in the number of CD3+ lymphocytes has been shown in patients with chronic lymphatic leukaemia. Recently, in vitro effects of the association on neutrophil function have been reported. In particular, the association was able to increase random migration, chemotaxis, phagocytosis-associated- and f-MLP-induced chemiluminescence. In this paper the authors evaluate the effects of L-lysine-L-arginine combination (lisargin) on several humoral and cell-mediated immunologic parameters in patients with recurrent infection. An increase of neutrophil random migration and chemotaxis (evaluated by a new technique, based on a computer assisted image processing system) was found. Furthermore an increase in the absolute number of lymphocytes involved in cytotoxic activity and IgG levels was observed.

IgE modification of the specific antidermatophagoides during the first year of specific immunotherapy (SIT).

Six subjects (4 female, 2 male), aged from 16 to 25 years, presented with allergic rhinitis to Dermatophagoides mites and received SIT by the sub-cutaneous route with delayed-release alpha fraction Bayropharm at the standard doses. Diagnosis was based on clinical history, skin tests and measurement of specific IgE at 0, 3, 9, and 12 months, by the fluoro-enzymatic technique (FAST). For comparison, in a reference group (n = 20) the IgE varied between 0.32 and 0.11 IU/ml for D1 and 0.31 to 0.09 IU/ml for D2. The eight patients had specific IgE titres of D1 = 0.96, D2 = 0.99. For these authors, the FAST technique used for the measurement of specific IgE, although less sensitive than the RIA technique of RAST, gives a good evaluation of SIT.