Abstract To elucidate the effects of inflammation on sperm quality, this study analysed classical sperm characteristics, leukocytes and elastase in neat semen, and sperm apoptotic markers, i.e. ...changes in plasma membrane phospholipid asymmetry, mitochondrial membrane potential (MMP), DNA integrity and intracellular reactive oxygen species (ROS), in semen prepared by density gradient using flow cytometry from 348 men of infertile couples. Increased leukocytes (⩾0.1 × 106 /ml) were associated with a decreased sperm concentration, motility and normal morphology ( P ⩽ 0.001). Sperm necrosis and DNA denaturation were increased (31.3 versus 26.6%, P = 0.020; 15.5 versus 11.5%, P = 0.011, respectively), whereas spermatozoa with normal MMP were decreased (64.1 versus 70.0%, P = 0.004). High leukocyte levels (⩾1 × 106 /ml) were not associated with any of the observed sperm parameters. At low elastase concentration (100–290 μg/l), DNA denaturation was higher (16.1 versus 10.5%, P = 0.024) compared with very low elastase concentration (<100 μg/l). A high elastase concentration (290–1000 μg/l) was associated with higher ROS index compared with low elastase concentration (1.28 versus 1.01, P = 0.016). Slightly increased leukocytes and elastase are associated with slightly poorer sperm characteristics and/or increased sperm necrosis, DNA denaturation and intracellular ROS and decreased MMP.
Summary Objective The aim of our prospective study was to test a specific T cell response to Helicobacter pylori before therapy and compare it to the success of H. pylori eradication 12 months later. ...Methods A total of 14 dyspeptic patients and 10 patients with previous H. pylori eradication failure were recruited into the study; before therapy their gastric samples for H. pylori cultivation and blood samples for dendritic cell cultivation were obtained. H. pylori antigens were produced to prime dendritic cells for stimulation of T lymphocyte response. Results The level of cytokine response by T cells was measured and results were compared with the success of H. pylori eradication one year later. There was a significantly increased response in expression of IFN-γ and IL-4 molecules by DCs stimulated T cells in subjects that successfully eradicated H. pylori compared with those who failed to eradicate the infection. Our results support the hypothesis that successful H. pylori eradication requires established anti- H. pylori immune response besides antibiotic treatment. Conclusion Effective IFN-γ cytokine response to H. pylori antigens seems to be of particular importance. Immunisation could be therefore beneficial for H. pylori eradication, while immunodeficiency could cause the failure in H. pylori eradication.
The objective of this study was to determine whether phagocytic activity in blood and proliferation of peripheral blood lymphocytes are impaired during perinatal period. The study comprised 18 ...primiparous sows (Landras × Large White) free from clinical signs of diseases. During the experiment blood samples were collected three times from each sow. Sampling was performed on three different dates, always from all sows at once. At the first date of sampling sows were 21 ± 3 days before parturition, at the second date ± 1 day around parturition time and at the third date 21 ± 3 days after parturition. Phagocytic activity of monocytes and granulocytes was assessed in heparinized whole blood with addition of fluorescein isothiocyanate (FITC)-labelled opsonized bacteria Escherichia coli and the percentage of phagocytes which have ingested bacteria was measured as fluorescence activity by flow cytometry. The percentage of phagocyting monocytes and granulocytes was lowest at parturition (72.6 ± 16.37, 52.4 ± 20.59) and significantly increased within the next 21 ± 3 days (86.5 ± 6.16, 69.89 ± 5.80). Similarly, the phytohemagglutinin (PHA) (10 μg/ml) stimulated in vitro lymphocyte response was suppressed by parturition in primiparous sows (p < 0.001).
Stefin B (cystatin B) is an inhibitor of lysosomal cysteine cathepsins and does not inhibit cathepsin D, E (aspartic) or cathepsin G (serine) proteinases. In this study, we have investigated ...apoptosis triggered by camptothecin, staurosporin (STS), and anti-CD95 monoclonal antibody in the thymocytes from the stefin B-deficient mice and wild-type mice. We have observed increased sensibility to STS-induced apoptosis in the thymocytes of stefin B-deficient mice. Pretreatment of cells with pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone completely inhibited phosphatidylserine externalization and caspase activation, while treatment with inhibitor of calpains- and papain-like cathepsins (2
S,3
S)-
trans-epoxysuccinyl-leucylamido-3-methyl-butane ethyl ester did not prevent caspase activation nor phosphatidylserine exposure. We conclude that sensitization to apoptosis induced by STS in thymocytes of stefin B-deficient and wild-type mice is not dependent on cathepsin inhibition by stefin B.
The immunological status before and after a comprehensive rehabilitation program was studied. Seven persons (4 males, 3 females, mean age 71.4 years) after lower limb amputation due to peripheral ...arterial disease (PAD) were subject to standard comprehensive rehabilitation program for amputees of four-week duration, which included training in activities of daily living, daily exercise of various types, training of crutch-assisted gait and use of leg prosthesis, and mild transcutaneous electrical stimulation. Before and after rehabilitation, peripherial blood was collected and the number and ratio of white blood cells were determined and analysed for the expression of cell surface antigens (CD3, CD4, CD8, CD19, CD25, CD69), cytokines (IFN-gamma, IL-4) and phagocytosis/oxidative killing functional tests. Due to strict patient selection criteria excluding serious accompanying disease, immunological parameters were within normal limits already before rehabilitation. After rehabilitation, an increase in oxidative burst was observed in monocytes and neutrophil granulocytes, but statistically significant only in monocytes. The expression of CD69 molecules by T cells and monocytes was significantly increased, as well as the expression of IL-4 by T cells. A significant decrease in the ratio of CD4 to CD8 cells was also found, but not a clinically critical one. It can therefore be concluded that the comprehensive rehabilitation treatment in patients with lower limb amputation due to PAD led to some prevailingly positive immunological changes, which were consistent with the patients' improved physical condition and clinical status. Reprinted by permission of Croatian Anthropological Society and Institute for Anthropological Research, Zagreb, Croatia
Actin nucleators initiate formation of actin filaments. Among them, the Arp2/3 complex has the ability to form branched actin networks. This complex is regulated by members of the Wiscott-Aldrich ...syndrome protein (WASp) family. Polymerization of actin filaments can be evaluated through flow cytometry by fluorescent phalloidin staining before and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). We identified a missense mutation in the gene ARPC1B (Arp2/3 activator subunit) resulting in defective actin polymerization in four patients (three of them were related). All patients (1 male, 3 female) developed microthrombocytopenia, cellular immune deficiency, eczema, various autoimmune manifestations, recurrent skin abscesses and elevated IgE antibodies. Besides four patients with homozygous mutation in ARPC1B, we also identified six heterozygous carriers without clinical disease (3 males, 3 females) within the same family. We developed a functional test to evaluate Arp2/3 complex function, which consists of flow cytometric detection of intracellular polymerized actin after
fMLP stimulation of leukocytes. Median fluorescence intensities of FITC-phalloidin stained actin were measured in monocytes, neutrophils and lymphocytes of patients, carriers, and healthy control subjects. We detected non-efficient actin polymerization in monocytes and neutrophils of homozygous patients compared to carriers or the healthy subjects. In monocytes, the increase in median fluorescence intensities was significantly lower in patients compared to carriers (104 vs. 213%;
< 0.01) and healthy controls (104 vs. 289%;
< 0.01). Similarly, the increase in median fluorescence intensities in neutrophils was significantly increased in the group with carriers (208%;
< 0.01) and healthy controls (238%;
< 0.01) and significantly decreased in the patient's group (94%). Our functional fMLP/phalloidin test can therefore be used as a practical tool to separate symptomatic patients from asymptomatic mutation associated to actin polymerization.
In the present study, we longitudinally monitored leukocyte subsets, expression of neutrophil surface adhesion molecules (CD62L and CD11b) and serum analytes in therapy-naïve patients with active ...giant cell arteritis (GCA). We collected blood samples at the baseline, and at weeks 1, 4, 12, 24, and 48 of follow-up, and evaluated short- and long-term effects of glucocorticoids (GC) vs. GC and leflunomide. Our aim was to identify candidate biomarkers that could be used to monitor disease activity and predict an increased risk of a relapse. Following high doses of GC, the numbers of CD4+ T-lymphocytes and B-lymphocytes transiently increased and then subsided when GC dose tapering started at week 4. In contrast, the numbers of neutrophils significantly increased during the follow-up time of 12 weeks compared to pre-treatment time. Neutrophil CD62L rapidly diminished after initiation of GC therapy, however its expression remained low at week 48, only in patients under combinatorial therapy with leflunomide. Levels of acute phase reactant SAA and IL-6 decreased significantly after treatment with GC and leflunomide, while levels of IL-8, IL-18, and CHI3L1 did not change significantly during the follow-up period. CHI3L1 was associated with signs of transmural inflammation and vessel occlusion and might therefore serve as a marker of fully developed active GCA, and a promising therapeutic target. Patients with relapses had higher levels of IL-23 at presentation than patients without relapses (
= 0.021). Additionally, the levels of IL-23 were higher at the time of relapse compared to the last follow-up point before relapse. IL-23 might present a promising biomarker of uncontrolled and active disease and could give early indication of upcoming relapses.
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