Expression of CD11a in lymphocyte subpopulation in immune thrombocytopenia.

Recent research demonstrates that the underlying mechanism in immune thrombocytopenia (ITP) is very complex. Lymphocyte function associated antigen-1 (LFA-1) plays important roles in autoimmune diseases. The purpose of this study was to investigate the expression of CD11a on lymphocytes and explore its possible role in ITP. The expression of CD11a on lymphocyte subpopulations (CD3(+) T cells, CD3(+)CD4(+) T cells, CD3(+)CD4(-) T cells, CD4(+)Foxp3(+) T regulatory cells and CD19(+) B cells) were analyzed by flow cytometry. Specific anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were assayed by modified monoclonal antibody specific immobilization of platelet antigens (MAIPA). The mean fluorescence intensity of CD11a on CD3(+) T, CD3(+)CD4(-) T and CD19(+) B lymphocytes were increased in ITP patients compared to healthy controls. No significant difference of CD11a expression on CD3(+)CD4(+) T cells or CD4(+)Foxp3(+) T regulatory cells was found between ITP patients and controls. Our data indicates the possible role of CD11a in the pathogenesis of ITP.

The effect of Na-selenite treatment on the oxidative stress-antioxidants balance of multiple organ failure.

PURPOSE: Our study tested the hypothesis that sodium (Na)-selenite expression treatment can reduce oxidative stress and increase plasma antioxidants, whereas modulating white blood cell antigen expression in severe sepsis. Selenite is a well known cofactor of glutathione peroxidases and other antioxidant enzymes; therefore, one may expect an antioxidant effect of treatment. MATERIALS: We randomized 40 severe septic patients into treatment and control groups. Treatment group (n = 21) received 1000-µg/2 hours Na-selenite load, followed by a 1000-µg/die medication. Oxidative stress markers, including malondialdehyde, maximal free radical production, and plasma antioxidants: free sulfhydryl groups, glutathione levels, and superoxide dismutase and catalase enzyme activity were measured. RESULTS: According to our results, the treatment regime successfully restored serum selenium levels. Treatment group developed a significant malondialdehyde increase by the fifth study day, whereas reactive oxygen species production decreased significantly. Reduced glutathione and plasma sulfhydryl groups showed no significant difference. Treatment group showed deteriorated expression of CD11a and slight increase of CD49d expression on monocytes throughout our study. CONCLUSIONS: Although our Na-selenite treatment regime successfully restored the selenium deficiency of severe septic patients, antioxidant and white blood cell antigen expression modulating effect of the therapy was not observed in our patient group.

Natalizumab treatment alters the expression of T-cell trafficking marker LFA-1 α-chain (CD11a) in MS patients.

OBJECTIVE: To determine the long-term effect of natalizumab (NTZ) treatment on the expression of integrins and chemokine receptors involved in the migration of T cells towards the central nervous system (CNS). METHODS: We drew the blood of 23 patients just before starting NTZ therapy and every 12 months thereafter, for up to 48 months of treatment. We assessed the ex-vivo expression of phenotype markers (CCR7 and CD45RA), CNS-addressing integrins (CD11a, CD49d and CD29) and chemokine receptors (CXCR3 and CCR6) in CD4+ or CD8+ T-cell subsets by flow cytometry. RESULTS: As compared to the pre-NTZ values, there was a marked increase in central memory (CCR7+/CD45RA-) CD4+ T cells and in effector memory (CCR7-/CD45RA-) CD8+ T cells at 12 and 24 months. In addition to an expected downregulation of both VLA-4 subunits (CD49d/CD29), we also found decreased T-cell expression of CXCR3 at 12 months, and of CD11a (LFA-1 αL subunit) at 12 months, but mostly at 24 months of NTZ treatment. CONCLUSION: Our data show a nadir of CD11a expression at 2 years of NTZ treatment, at the peak of incidence of progressive multifocal leukoencephalopathy (PML), indirectly suggesting that a lack of these molecules may play a role in the onset of PML in NTZ-treated patients.

The effects of arterial blood pressure reduction on endocan and soluble endothelial cell adhesion molecules (CAMs) and CAMs ligands expression in hypertensive patients on Ca-channel blocker therapy.

BACKGROUND/AIMS: To determine the effect of arterial blood pressure (BP) reduction on endocan and soluble cell adhesion molecules' (sCAM) plasma concentration and expression of their ligands on circulatory leukocyte subpopulations. METHODS: 24 hypertensive subjects of both sexes (age: 53±8 yrs) were treated with Ca-channel blocker, amlodipin (5-10 mg/day for 8 weeks; to reach BP≤139/89mmHg). The serum sCAMs and endocan concentrations were determined by ELISA kits. Level of ICAM/VCAM ligands on leukocytes was assessed by flow cytometry. Paired t-test, or t-test were used as appropriate, with Pearson's correlation calculated; p<0.05 was considered significant (SigmaPlot v.11). RESULTS: sICAM-1 and sVCAM-1 were decreased (p≤0.001 and p=0.002, respectively), while E-selectin concentration was increased after amlodipin treatment (P=0.014). CD11a/LFA-1 (ICAM-1 and endocan ligand) was significantly increased in all three cell types with BP decrease. CD15 and CD49d/VLA-4 (VCAM-1 ligand) did not change after the treatment. There was significant positive correlation of systolic and diastolic BP with ICAM-1 and VCAM-1, and significant negative correlation of systolic BP with CD11a/LFA-1. Endocan significantly positively correlated with ICAM-1. CONCLUSIONS: The increased expression of ICAM/VACM ligands, together with decrease of sCAMs and endocan suggests the de-activation of endothelium with reduction in BP, decreasing the adherence of circulatory leukocytes to endothelium; subsequently decreasing the risk for development of atherosclerosis.

Blast cell deficiency of CD11a as a marker of acute megakaryoblastic leukemia and transient myeloproliferative disease in children with and without Down syndrome.

BACKGROUND: The classification of acute myeloid leukemia (AML) FAB subtype M7 relies on immunophenotypic assessment. CD41 is expressed throughout all stages of maturation of megakaryocytes and has therefore been described as a specific blast cell marker in AML M7 as well as in transient myeloproliferative disease (TMD) of patients with Down syndrome (DS). However, technical difficulties underlie the need for new markers for these entities. METHODS: We evaluated the expression of human lymphocyte function-associated antigen 1 (CD11a) in a large cohort of pediatric AML and TMD patients (n = 91) of the Austrian AML-BFM 98 and 2004 studies. RESULTS: We found a consistent deficiency of CD11a as assessed by mean fluorescence intensity in all patients with non-DS AML M7 (n = 8) and M6 (n = 1), all cases of classical DS-AML (n = 12) as well as TMD (n = 15) that was statistically significant in comparison to non-DS AML M0-M5 patients (n = 55; P < 0.001, sensitivity 100%). Only three of 55 Non-DS M0-5 patients were CD11a deficient (specificity 95%). Monocytic leukemias (M4/5) and normal monocytes typically showed a high CD11a expression, FAB types M1/2 and normal neutrophils an intermediate expression level, while all M3 leukemias were rather low in CD11a expression. CONCLUSIONS: We conclude, that deficiency of CD11a expression should be added to the diagnostic criteria of AML-M7, classical DS-AML and TMD.

Effects of CD70 and CD11a in immune thrombocytopenia patients.

INTRODUCTION: CD70 and CD11a are co-stimulatory molecules that are important for the immune functions of T, B lymphocytes. Over-expressions of CD70 or CD11a cause T cell to be autoreactive. OBJECTIVES: The purpose of this study was to explore the effect of CD70 and CD11a in immune thrombocytopenia (ITP). METHODS: CD70 and CD11a mRNAs and protein expressions in CD4(+) T cells from ITP patients were measured respectively by real-time quantitative-PCR (RT-PCR) and flow cytometry. The apoptosis of T cells, B cells, and platelets in the PBMCs were analyzed by flow cytometry, and secretion of IL-4, IFN-γ, as well as IgG in the reaction supernatant were detected by ELISA. In order to investigate the effects of CD70 and CD11a over-expression on pathogenesis of ITP, anti-CD70, and anti-CD11a mAbs were used to block the signaling pathways. RESULTS: CD70 and CD11a mRNAs and protein expressions in CD4(+) T cells from ITP patients were significantly higher than healthy controls. In vitro co-culturing of PBMCs with anti-CD70 or anti-CD11a, the apoptosis of T, B lymphocytes were significantly increased but apoptosis of platelets were reduced. Anti-CD11a and anti-CD70 both significantly suppressed the secretion of IFN-γ, while anti-CD11a significantly promoted the secretion of IL-4. There was no significant difference in the healthy group. CONCLUSIONS: CD70 and CD11a facilitate the survival of T, B lymphocytes and indirectly enhance the destruction of platelets in ITP. Blockade of CD70 or CD11a are promising therapeutic approaches for ITP.

Aggressive and chronic periodontitis correlate with distinct cellular sources of key immunoregulatory cytokines.

BACKGROUND: Chronic periodontitis (CP) and aggressive periodontitis (AP) are inflammatory diseases and the main cause of dental loss in adults. We aimed to investigate the expression of adhesion molecules and the source of proinflammatory and anti-inflammatory cytokines in circulating mononuclear cells from patients with CP and AP. METHODS: Peripheral blood mononuclear cells from healthy controls and CP or AP patients were collected. The expression of the cell adhesion molecules CD11a and CD11b, and the cellular sources of interleukin (IL)-4, IL-10, IL-12, interferon-γ, and tumor necrosis factor-α by distinct subpopulations of circulating leukocytes were determined using flow cytometry. RESULTS: The expression of CD11a, but not CD11b, was significantly higher within the CD4(+) and CD8(+) T cells in CP and AP than in healthy controls. The frequencies of tumor necrosis factor-α-expressing CD4(+) T cells and CD14(+) cells were higher in AP and CP, compared to healthy controls, respectively. Moreover, the frequency of IL-10 expressing CD14(+) cells was higher in CP, but not AP, compared to healthy controls CD4(+) T cells committed to IL-4 production was higher in CP than in healthy controls. CONCLUSION: These results suggest the participation of CD11a in the pathogenesis of periodontal lesions and show distinct cellular sources of immunoregulatory cytokines in AP versus CP.

[Changes in oxidative stress and hemostatic parameters in the course of thrombolytic therapy of pulmonary embolism]. / Az oxidatív stressz és haemostasis paramétereinek változása életveszélyes tüdoembólia thrombolyticus kezelése során.

UNLABELLED: Acute pulmonary embolism is the third most common cause of cardiovascular mortality. Thrombolytic treatment of massive pulmonary embolism can be complicated with haemorrhage, re-thrombosis and oxidative stress. AIMS: The purpose of this study was to evaluate the changes in platelet aggregation, haemostatic, leukocyte function parameters and oxidative stress in patients with acute pulmonary embolism treated with thrombolytics. METHODS: Fifteen patients undergoing thrombolysis with ultra-high dose streptokinase ( n = 8), or alteplase ( n = 7) treatment were studied. Arterial blood samples were taken before (baseline) and after thrombolysis between the 4th and 24th hour at every four hours, on the second day twice a day and daily on the 3rd, 4th, 5th and 30th day. Platelet aggregation was examined as spontaneous and induced aggregation with adrenaline, collagen and adenosine diphosphate. D-dimer and fibrinogen were measured 8 hourly on the first day and later at the same time intervals as above. To analyse oxidative stress, blood samples were collected prior to thrombolysis, and then 8 hours, 1, 3, 5 and 30 days after treatment. Malondialdehyde, reduced glutathion, plasma sulphydryl groups levels, superoxide dismutase and myeloperoxidase enzyme activities were measured in plasma or whole blood for monitoring of the oxidative stress markers. Production of reactive oxygen species in whole blood was measured by luminol dependent chemiluminescence. Flow cytometry was used to determine CD11a, CD18, and CD97 surface antigen expression on leukocytes. RESULTS: In streptokinase group, adrenaline induced platelet aggregation decreased at the 4th and 8th hour ( p < 0.03) and was significantly lower than in the alteplase group at the 36th hour and on the 3rd day. Platelet aggregation induced by adenosine diphosphate was lower at the 4th hour than at baseline in streptokinase group ( p < 0.05). Collagen induced platelet aggregation was lower at the 4th and 8th hour than at baseline ( p < 0.05) in streptokinase group. Compared to baseline, fibrinogen levels decreased in both groups after thrombolysis. D-dimer levels elevated significantly in both therapeutic groups at the 8th hour. Spontaneous platelet aggregation was not detectable and major bleeding or re-embolism was not documented. The elevated malondialdehyde, reactive oxygen species and myeloperoxidase, decreased reduced glutathion and plasma sulphydryl levels indicated the presence of oxidative stress in patients with pulmonary embolism. Malondialdehyde significantly increased, reduced glutathion significantly decreased following thrombolysis. Reactive oxygen species production peaked on the 3rd and 5th days. Thrombolysis was accompanied by significant decrease in granulocyte and monocyte CD11a and CD18 as well as in granulocyte CD97 expression ( p < 0.05). CONCLUSION: Massive/submassive pulmonary embolism and thrombolysis injures inducible platelet aggregation. The changes in fibrinogen levels correlate significantly with the improvement of pulmonary perfusion which shows the effect of thrombolysis. Pulmonary embolism induced oxidative stress was detected on patients before thrombolysis. Thrombolytic treatment of pulmonary embolism augmented the increase of oxidative stress response and leukocyte activation following reperfusion, and these parameters normalised only on the 30th day.

Effect of dietary fish oil supplementation on cellular adhesion molecule expression and tissue myeloperoxidase activity in diabetic mice with sepsis.

This study investigated the effect of n-3 fatty acids on adhesion molecules and tissue myeloperoxidase (MPO) activity in diabetic mice with sepsis. Diabetes was induced by a streptozotocin injection. Mice with blood glucose levels exceeding 2000 mg/l were considered diabetic. Diabetic mice were assigned to two groups with a medium-fat (10 %, w/w) diet either provided by soyabean oil (SO, n 30) or fish oil (FO, n 30). n-3 fatty acids provided 4.3 % of the total energy and the n-3/n-6 fatty acid ratio was 1:2 in the FO diet. After feeding the respective diet for 3 weeks, all mice had sepsis induced by caecal ligation and puncture (CLP) and were killed at 0, 6 or 24 h after CLP, with ten mice at each time-point. The result showed that compared with the SO group, FO group had lower PGE2 and TNF-alpha levels in peritoneal lavage fluid after CLP. Lymphocyte CD11a/CD18 expressions were higher at 6 h, whereas the percentage was lower at 24 h in the SO group than in the FO group. Neutrophil CD11b/CD18 expressions were significantly higher in the SO group than in the FO group at 0 h. The FO group had lower organ MPO activities at various time-points after CLP when compared with those of the SO group. The present findings suggest that compared with the diabetic mice fed SO, a low-dose n-3 fatty acid supplementation may attenuate leucocyte adhesion and infiltration into tissues in diabetic mice complicated with sepsis.

Detailed analysis of intrahepatic CD8 T cells in the normal and hepatitis C-infected liver reveals differences in specific populations of memory cells with distinct homing phenotypes.

In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7-, L-selectin-) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin- cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin- intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.

Quantitative expression of the major homing integrins alphaL and alpha4 on human cord blood hematopoietic progenitor and stem cells.

An increased traffic of hematopoietic progenitor cells (HPC) between bone marrow and peripheral organs is a peculiar feature of the allergic inflammation. It has been recently reported that the sublingual form of specific immunotherapy (SLIT) is capable of reducing such an increased HPC traffic. The House Dust Mite major antigen Der p1 has been proved to up-regulate the expression of the ICAM-1 and VCAM-1 endothelial addressins, supporting the view of an inflammatory cell recruiting at the site of allergen extract administration. In the present work we have investigated, by flow-cytometric techniques, the expression of the two major integrins CD11a (LFA-1) and CD49d (VLA-4) that are the homing receptor cognate for ICAM-1 and VCAM-1 on human cord blood CD34 hematopoietic progenitor and stem cells. Even if both the investigated molecules resulted detectable on CD34+ HPC surfaces, being the system redundant, the density of the cellular expression was significantly higher for CD49d (median value: 158) than CD11a (median value: 20.5), suggesting a preferential usage of the homing axis VLA-4/VCAM-1. Results consistency with outcomes of clinical trials that relate SLIT efficacy to allergen dosage is discussed.

Natural killer cell lytic activity and CD56(dim) and CD56(bright) cell distributions during and after intensive training.

The purpose of this study was to examine the impact of intensive training for competitive sports on natural killer (NK) cell lytic activity and subset distribution. Eight female college-level volleyball players undertook 1 mo of heavy preseason training. Volleyball drills were performed 5 h/day, 6 days/wk. Morning resting blood samples were collected before training (Pre), on the 10th day of training (During), 1 day before the end of training (End), and 1 wk after intensive training had ceased (Post). CD3(-)CD16(bright)CD56(dim) (CD56(dim) NK), CD3(-)CD16(dim/-)CD56(bright) NK (CD56(bright) NK), and CD3(+)CD16(-)CD56(dim) (CD56(dim) T) cells in peripheral blood were determined by flow cytometry. The circulating count of CD56(dim) NK cells (the predominant population, with a high cytotoxicity) did not change, nor did the counts for other leukocyte subsets. However, counts for CD56(bright) NK and CD56(dim) T cells (subsets with a lower cytotoxicity) increased significantly (P < 0.01) in response to the heavy training. Overall NK cell cytotoxicity decreased from Pre to End (P = 0.002), with a return to initial values at Post. Lytic units per NK cell followed a similar pattern (P = 0.008). Circulating levels of interleukin-6, interferon-gamma, and tumor necrosis factor-alpha remained unchanged. These results suggest that heavy training can decrease total NK cell cytotoxicity as well as lytic units per NK cell. Such effects may reflect in part an increase in the proportion of circulating NK cells with a low cytotoxicity.

Separation of CD34 positive cells and determination of surface homing antigen.

To study the differences in homing potential between bone marrow cells and umbilical blood cells, CD34 positive cells were obtained from bone marrow (BM) and umbilical blood (UB) by the direct cell separation with domestic immunomagnetic beads. The expression of the two adhesion molecules CD11a/CD18 and CD44 were examined. After separation, CD34 positive cells accounted for 51%-82% of the harvested cells and dye-resistance rate was 82%-88%. The expression of CD11a/CD18 and CD44 on the surfaces of UB cells was 49.6% +/- 10.2% and 37.7% +/- 10.3% respectively. On BM cells they were 50.2% +/- 6.2% and 34% +/- 13.3% respectively. There were no significant differences in the expression of these two molecules. It was concluded that the cell separation method with domestic immunomagnetic beads was effective and the stem cells from UB could serve as an alternative source for transplantation.

Genetic and immunological assessment of a bone marrow transplantation in a patient with a primary immune defect: leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) was suspected in a three weeks old girl from a family with an established history of LAD with a lack (less then 1%) of the beta 2 integrins CD 11a, b/CD 18 expression at the leukocytes surface, was engrafted with her mother HLA identical bone marrow at the age of 14 months. Repeated post transplantation (up to 22 months). Immunological assessments showed a good engraftment with 97% of the lymphocytes expressing CD11a/CD18. Cells proliferated normally in response to PHA and to Tetanus toxoïd after revaccination. The level of serum immunoglobulins was normal. Investigation of the CD18 intragenic polymorphic marker Avall before and after bone marrow transplantation (BMT) showed a transition from the Avall +/+ genotype to the mother's Avall +/- genotype. Similarly DNA fingerprints obtained with the patient genomic DNA, prepared from PBMC, prior and after transplantation, showed that the patient's DNA fingerprints pattern matched the mother's one. These findings are consistent with the good engraftment observed clinically. This study emphasizes the usefulness of the molecular techniques to evaluate the degree of chimerism in monitoring the outcome of bon marrow transplantation.