A pharmacodynamic comparison of 5 anti-platelet protocols in patients with ST-elevation myocardial infarction undergoing primary PCI.

BACKGROUND: Despite advances in anti-platelet treatments, there still exists an early increase in both ischemic as well as bleeding events following primary PCI in patients with ST-elevation myocardial infarction (STEMI). Platelet inhibition data of different anti-platelet treatments in the acute phase of a myocardial infarction might offer some insight into these problems. The aim of this study was to evaluate the pharmacodynamic profile of 5 different anti-platelet treatments in the acute phase of STEMI in patients undergoing primary PCI. METHODS: A total of 223 STEMI patients undergoing primary PCI were prospectively included. Patients received either pre-hospital clopidogrel only, pre-hospital clopidogrel followed by prasugrel switch in the cath lab, prasugrel treatment only, pre-hospital clopidogrel followed by ticagrelor switch in the cath lab or pre-hospital ticagrelor only. Platelet reactivity was measured serially using vasodilator-stimulated phosphoprotein (VASP). RESULTS: Patients receiving pre-hospital clopidogrel followed by prasugrel switch showed similar platelet inhibition data as patients receiving prasugrel only, with more than 90% being good responders the day after PCI. Average time from prasugrel administration to a VASP value of <50% was 1.5 hours. In patients receiving pre-hospital ticagrelor, 50% were good responders at completion of PCI and average time to a VASP-value of <50% was 2.3 hours. Only 32% of patients receiving clopidogrel only were responders the day after PCI. CONCLUSIONS: Switching from an upstream bolus dose of clopidogrel to prasugrel at the time of PCI, appeared as a safe and feasible option with no tendency for overshoot or attenuation of platelet inhibition. Pre-hospital administration of ticagrelor was associated with a 50% good responder rate at completion of PCI.

TLR3 in human eosinophils: functional effects and decreased expression during allergic rhinitis.

BACKGROUND/AIM: Viral respiratory infections are increasingly implicated in allergic exacerbations. Virus-induced activation of eosinophils through Toll-like receptors (TLRs) could be involved. The present study was designed to examine TLR3 expression in eosinophils from bone marrow (BM) and peripheral blood (PB) during symptomatic allergic rhinitis, and to evaluate the functional responsiveness of TLR3 in purified eosinophils. METHODS: BM and PB samples were obtained from healthy volunteers and patients with seasonal allergic rhinitis outside and during the pollen season. Eosinophils were analyzed for TLR3 expression by flow cytometry. Polyinosinic:polycytidylic acid [poly(I:C)], an agonist for TLR3, was used to assess its functional role in purified eosinophils and the intracellular signaling pathways involved. RESULTS: TLR3 expression was demonstrated in BM and PB eosinophils. It was higher in BM-derived than in circulating cells and it was downregulated in both compartments during symptomatic allergic rhinitis. TLR3 expression was also downregulated in the presence of interleukin (IL)-4 and IL- 5. Stimulation with poly(I:C) increased the percentage of CD11b+ cells and enhanced the secretion of IL-8, effects mediated via the p38 mitogen-activated protein kinases and nuclear factor-kappaB signaling pathways. Moreover, pretreatment with IL-5 augmented the poly(I:C)-induced IL-8 release. CONCLUSIONS: Eosinophils activated via TLR3 might be more able to home and recruit leukocytes to sites of inflammation. The decreased TLR3 expression during symptomatic allergic rhinitis and in the presence of Th2 cytokines indicates a role in allergic airway inflammation. Thus, eosinophils might function as a link between viral infections and exacerbations of allergic disease.

Expression of Toll-like receptor 9 in nose, peripheral blood and bone marrow during symptomatic allergic rhinitis.

BACKGROUND: Allergic rhinitis is an inflammatory disease of the upper airway mucosa that also affects leukocytes in bone marrow and peripheral blood. Toll-like receptor 9 (TLR9) is a receptor for unmethylated CpG dinucleotides found in bacterial and viral DNA. The present study was designed to examine the expression of TLR9 in the nasal mucosa and in leukocytes derived from different cellular compartments during symptomatic allergic rhinitis. METHODS: The study was based on 32 patients with seasonal allergic rhinitis and 18 healthy subjects, serving as controls. Nasal biopsies were obtained before and after allergen challenge. Bone marrow, peripheral blood and nasal lavage fluid were sampled outside and during pollen season. The expression of TLR9 in tissues and cells was analyzed using immunohistochemistry and flow cytometry, respectively. RESULTS: TLR9 was found in several cell types in the nasal mucosa and in different leukocyte subpopulations derived from bone marrow, peripheral blood and nasal lavage fluid. The leukocyte expression was generally higher in bone marrow than in peripheral blood, and not affected by symptomatic allergic rhinitis. CONCLUSION: The widespread expression of TLR9 in the nasal mucosa along with its rich representation in leukocytes in different compartments, demonstrate the possibility for cells involved in allergic airway inflammation to directly interact with bacterial and viral DNA.

A high frequency of MDSCs in sepsis patients, with the granulocytic subtype dominating in gram-positive cases.

The causative microorganisms dictate the type of MDSC generated in sepsis patients, and a large proportion of PMN-MDSCs in gram-positive sepsis includes immunosuppressive myeloid blasts. MDSCs constitute a heterogeneous population of immature myeloid cells that potently suppress immune responses. They were identified originally in cancer patients and have since been reported to occur also in chronic inflammation, autoimmunity, and even bacterial infections. Human MDSCs are commonly divided into Mo-MDSCs and granulocytic (PMN-MDSCs) subtypes. To what extent the bona fide cancer MDSCs are representative of the proposed MDSCs found in other diseases is not well known. PMN-MDSCs have been found previously to be enriched among LDGs in density gradient-centrifuged blood. In this study, we analyzed potential MDSCs in sepsis patients with different causative microorganisms, using total peripheral blood compared with density gradient-centrifuged blood. We found a high frequency of typical CD14(+)HLA-DR(low) Mo-MDSCs in all sepsis patients, whereas the typical PMN-MDSCs, as well as a prominent CD14(low) PMN-MDSC-like population, appeared preferentially in gram-positive cases. The CD14(low) PMN-MDSC variant was demonstrated to suppress T cell proliferation in vitro via a ROS-dependent mechanism, to display an increased IL-10:TNF-α ratio, and to present with signs of immaturity: blast morphology and low cytokine levels. We conclude that a spectrum of cells with MDSC features is enriched in sepsis and that the microbial origin of sepsis contributes to the substantial interindividual patient variation in the MDSC pattern.

The activation pattern of blood leukocytes in head and neck squamous cell carcinoma is correlated to survival.

Head and neck squamous cell carcinoma (HNSCC) is known to cause substantial immunosuppression. The present study was designed to characterize blood leukocyte activation in HNSCC and to investigate if the individual activation pattern could be related to tumor progress and survival. The leukocyte activation profile of HNSCC patients and healthy controls was assessed with flow cytometry. HNSCC patients displayed increased numbers of monocytes, neutrophils and total leukocytes as well as an enhanced neutrophil/lymphocyte ratio. In addition, patients had a higher percentage of CD69(+), CD71(+) and CD98(+) T cell subsets and NK cells, and a reduced expression of L-selectin in CD14(high)CD16(+) monocytes and neutrophils, when compared to controls. These changes could be correlated to both tumor burden and spread to lymph nodes. Among the cancer patients an increased neutrophil/lymphocyte ratio, a low neutrophil and CD14(high) CD16(+) monocyte activation state and an elevated CD4/CD8 ratio were related to poor survival. In contrast, a high percentage of CD98(+) Th cells appeared to be associated with a better outcome. Taken together, the present data indicate that HNSCC causes activation of blood leukocytes and that the individual activation pattern can be linked to prognosis.

Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer.

BACKGROUND: Flow cytometry allows the use of several antibodies in addition to light scatter, and most flow cytometers will provide at least seven measurements on each cell passing through the laser beam. A skilled microscopist will classify at least 14 cell classes in bone marrow or blood. Our goal was to use the seven parameters available in our flow cytometer to provide a reliable differential count using only one tube. METHODS: Peripheral blood samples were analyzed on the Beckman Coulter LH750 cell counter, and the flagging and messages from the cell counter were used to select normal or pathological samples. Samples without flags (N = 50), with >2% erythroblasts (N = 80), or with "Blast" or "Verify diff" flags (N = 54) were investigated. We used a lyse-no-wash method to ensure minimal loss of fragile cells with live gating on DRAQ5-positive cells to acquire only nucleated cells. The FL-1 to FL-4 channels were used for the antibodies CD36-FITC, CD203-PE, CD138-PE, CD45-ECD, CD16-Pcy5, and CD56-Pcy5. FL-5 was used for the DNA-stain DRAQ5. RESULTS: Using live gate acquisition on DRAQ5, we were able to classify total nucleated cells into 10 classes. We were unable to identify megakaryocytes, but platelets could be studied by rerunning the sample after dilution and gating on DRAQ5-negative CD36-posive events. Validation against digitized microscopy and cell counter showed linear correlations within each cell class with correlation coefficients that seem reasonable for cellular classification. The lowest correlation was found for basophil granulocytes. Flow cytometry detected twice as many immature neutrophils compared to microscopy. CONCLUSIONS: We have designed a one-tube immunophenotyping panel for classification of total nucleated cells and platelets in blood or bone marrow. The seven parameters available in one single tube in our cytometer seem to be enough for reliable differential count even in difficult pathological samples. The analytical imprecision of the flow cytometer differential was much lower than that obtained with microscopy or cell counter differentials.

Quantitation of repetitive epitopes in glycosaminoglycans immobilized on hydrophobic membranes treated with cationic detergents.

Glycosaminoglycans (GAGs) are linear carbohydrate polymers containing repetitive sequences of differently sulfated uronic acid and glycosamine residues that are recognized by antibodies raised against proteoglycans. We have developed a method to demonstrate such repetitive sequence motifs in isolated GAG chains immobilized on hydrophobic membranes derivatized with cationic detergents. Six monoclonal antibodies directed against Cs (2B6, 3B3, Cs56, and 1B5), Hs (HepSS), and Ks (5D4) were used to detect native and chondroitinase-generated epitopes in the immobilized GAGs. All antibodies, except 1B5, were able to detect epitopes in both proteoglycans and isolated GAGs. Type of detergent and buffer composition affected the accessibility and the retention of immobilized GAGs. The epitope density, i.e., the number of repetitive epitopes per GAG mass, was estimated as the ratio between antibody (epitope) and Alcian blue (mass) staining measured simultaneously. The epitope profiles, using six antibodies, were different for each sample (CsA, CsC, Ds, Hs, intact cartilage, and human serum). The epitope profile may be used as a structural characteristic of a GAG population. Electrophoretic separation of GAGs based on their glucuronic/ioduronic acid content and O-sulfate/N-sulfate ratio was performed using a diethylene glycol-diaminobutanol agarose gel. The electrophoretic populations were characterized by immunoblotting to detergent-treated membranes.