Reliable data on plasminogen activator (PA) activities in blood of patients receiving fibrinolytic treatment are lacking. This is due to the continuing in vitro action of PA after blood withdrawal. We have elaborated a new simple stabilization technique for plasma involving the addition of arginine in final concentrations greater than 500 m M. In this study, new assays for PA in stabilized plasma are developed. The assay was performed with substrate plasma, that is, pooled normal plasma, preoxidized with chloramine-T; oxidant amount and oxidation time were optimized. The chloramine consumption by plasma was assayed with a KJ-assay (absorbance increase at 405 nm by addition of 200 μL 4 M KJ to 25 μL oxidized plasma). The substrate plasma concentration in the PA assay and the PA acting time was optimized. The inhibition of PA by the cations Na + , K + , Mg 2+ , and Ca 2+ was evaluated. The optimized PA assay consists of incubation of 10 μL arginine-stabilized plasma with 10 μL 1.5 M arginine, pH 8.7 and 10 μL 100 m M CT in PBS. After 30 minutes (37°C), 175 μL 15 m M CT oxidized EDTA plasma are added. After 40 minutes (37°C), 75 μL Stop-CS Reagent is added and ΔA at 405 nm was determined, giving PA + plasmin activity in plasma. A control value (basal plasmin activity) consists of the addition of Stop-CS Reagent before 175 μL oxidized EDTA plasma. To obtain plasmatic PA activity, the control value has to be subtracted from the PA main value. The assay is matrix-independent and linear up to 1250 IU/mL t-PA, 790 U/mL reteplase, or 199 IU/mL u-PA (37 n M). With arginine stabilization of plasma and the described determination of plasminogen activator activity in arginine-stabilized plasma, it is feasible to determine the activity of plasminogen activators in blood of patients receiving fibrinolytic treatment without artefactual in vitro changes of the samples.
<b><i>Background:</i></b> Exposure to microbes and their components may affect the maturation of the immune system.<b> </b>We examined the association of house dust microbial content with cytokine-producing capacity at birth and at the age of 1 year. <b><i>Methods:</i></b> Production of TNF-α, IFN-γ, IL-5, IL-8 and IL-10 at birth (n = 228) and at the age of 1 year (n = 200) following 24- and 48-hour whole-blood stimulation with staphylococcal enterotoxin B (SEB), lipopolysaccharide and the combination of phorbol ester and ionomycin was measured. Concentrations of ergosterol (marker for fungal biomass), muramic acid (marker for Gram-positive bacteria) and 3-hydroxy fatty acids with a carbon chain length from 10 to 14 (marker for Gram-negative bacteria) in living room floor dust were analyzed using gas chromatography-tandem mass spectrometry. Five single microbial species or groups were determined using a quantitative polymerase chain reaction method. <b><i>Results:</i></b> A high total level of the studied Gram-positive bacteria in general or <i>Mycobacterium </i>spp. in house dust was associated with decreased SEB-stimulated IFN-γ production, especially at the age of 1 year. The total level of indoor fungi analyzed <i>(Penicillium</i> spp<i>., Aspergillus </i>spp<i>. </i>and<i> Paecilomyces variotii</i> group, <i>Trichoderma viride/atroviride/koningii,</i><i>Wallemia sebi)</i> was also inversely associated with IFN-γ production at the age of 1 year, but this association did not remain significant after adjustment for potential confounders. A few associations were found between microbial exposures and other measured cytokines. <b><i>Conclusions:</i></b> High indoor microbial exposures may affect immune development in early life by reducing T helper type 1 cytokine secretion capacity. The observed hyporesponsiveness may reflect the adaptation of the immune system to environmental antigens. In future, more attention should be paid especially to the immunomodulatory role of exposures to Gram-positive bacteria.
The main function of neutrophils is to provide a front line of defense against invasive bacteria. Disturbances in the functioning of neutrophils lead to repeated and life-threatening infections caused by bacteria and fungi (1)(2). Pathological neutrophil functions are detected as permanent inborn metabolic defects of NADPH oxidase with oxidative burst [chronic granulomatous disease (CGD) (2)(3)], glutathione peroxidase (4), and adhesion molecules (2)(5). Moreover, transient disturbances of phagocytosis may be detected in systemic infections (5)(6)(7)(8)(9), acute pancreatitis (10), tuberculosis (11), and Wegner granulomatosis (12), as well as under special conditions such as in newborns (6)(13)(14), very old persons (15), or in persons undergoing therapies with cytokines, prednisolone, or anesthetics (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(14)(16).
The following clinical and laboratory findings indicate that assessment of granulocyte function is needed: increased susceptibility to bacterial infections, therapy-resistant infections, recurrent infections with nonpathogenic microorganisms, lymphadenitis, abscesses of liver or lung, osteomyelitis, recurrent stomatitis, or gingivitis. Granulocytopenia and defects of B cells or complement compartment must be excluded (17). Phagocytosis, adhesion molecules CD18 and CD11b for leukocyte adhesion defect I or CD15s for leukocyte adhesion defect II, and production of oxygen radicals upon stimulation for CGD can be tested by flow cytometric determinations. Disturbances such as Chediak-Higashi syndrome, hyper IgE syndrome, or glycogenesis type Ib need other techniques.
One of the most common inherited granulocyte defects is CGD. The nitroblue tetrazolium dye reduction assay, the gold standard for diagnosis of CGD in the past (18)(19)(20), has been replaced to a large extent by flow cytometry-based procedures (18)(21)(22)(23). Commercially available …
