Emel Demiralp

Analysis of pleural effusions using flow cytometry.

Flow cytometry allows a rapid and accurate analysis of the cells in serous fluids. The aim of this study was to evaluate the use of flow cytometric analysis in malignant pleural effusions. 26 patients (13 females, 13 males; mean age 52 +/- 19 years; range 16-82) were included in the study. 15 had malignant pleural effusions (7 adenocarcinoma, 2 lymphoma, 2 chronic myeloid leukemia, 1 ovarian carcinoma, 1 small cell lung carcinoma, 1 squamous cell lung carcinoma and empyema, and 1 malignant mesothelioma) with positive cytology. 2 had benign effusions associated with malignancy (1 squamous cell lung carcinoma and congestive heart failure, and 1 neuroblastoma and hypoproteinemia). 9 had benign effusions (3 tuberculosis, 1 congestive heart failure, 3 parapneumonic pleural effusion, 1 benign mesothelioma, and 1 pulmonary embolism). Flow cytometric analysis of pleural effusions revealed an increased DNA index in malignant effusions: 1.32 +/- 0.44 versus 0.88 +/- 0.23 in benign effusions (p < 0.04). The cell cycle distribution of cells such as G1/G0 and S in malignant effusions did not differ from that of benign pleural effusions; however G2+M increased significantly in malignant effusions (p < 0.03). Using analysis of mononuclear immunophenotyping, CD3+, CD4+, and CD8+ cells did not show any significant difference between the two groups. The lymphocyte activation marker CD38 was positive in 57.6 +/- 11.5% of malignant fluid cells and 38.5 +/- 6.2% of benign fluid cells (p < 0.04). The mean carcinoembryonic antigen levels in malignant and benign pleural effusions were 98.7 +/- 157.3 and 0.9 +/- 1.2 ng/ml, respectively (p < 0.03). In conclusion, the results of our study indicate that finding cells with an abnormal DNA content strongly supports the diagnosis of malignant pleural effusions. Additionally, mononuclear cell phenotypes have to be taken into consideration for malignant pleural effusions, particularly activated T cells. We recommend that flow cytometry should be performed if the cytology is equivocal.

Use of T Cell-Based Diagnosis of Tuberculosis Infection to Optimize Interpretation of Tuberculin Skin Testing for Child Tuberculosis Contacts

BACKGROUND Treatment of recent tuberculosis infection in children aged <2 years is essential, because of high risk of progression to disease, but diagnosis is hindered by the inaccuracy of the tuberculin skin test (TST). More-accurate T cell-based tests of infection could enhance diagnosis by optimizing interpretation of the TST results. METHODS A total of 979 child tuberculosis contacts in Istanbul underwent the TST and enzyme-linked immunospot assay. Using enzyme-linked immunospot test results as a reference standard, we assessed the effect of age and bacille Calmette-Guérin (BCG) vaccination on the sensitivity and specificity of the TST, and we computed the optimal TST cutoff points, using receiver operating characteristic curves. RESULTS With a TST cutoff point of >or=10 mm, the sensitivity of the TST was 66% for children aged <2 years, which was lower than that for older children (P= .006). Specificity was 75% for BCG-vaccinated children, compared with 92% for unvaccinated children (P= .001). Optimal cutoff points improved TST specificity for children with 1 BCG scar, with little loss of sensitivity. Despite the use of optimal cutoff points, TST sensitivity remained <70% for children aged <2 years, specificity remained <87% for BCG-vaccinated children aged >or=2 years, and overall accuracy was low for children with >1 BCG scar. CONCLUSIONS Negative results of the TST cannot exclude tuberculosis infection for child tuberculosis contacts aged <2 years, which supports the use of preventive therapy regardless of the TST results for this age group. In children aged >or=2 years, the accuracy of the TST can be improved by adjustment of cutoff points for BCG-vaccinated children but remains poor for children with >1 BCG scar. This methodology can define optimal TST cutoff points for diagnosis of tuberculosis infection tailored to target populations.

Effects of coronary artery bypass grafting on cellular immunity with or without cardiopulmonary bypass: changes in lymphocytes subsets

Cell-mediated immunity responses decrease after all kinds of surgical procedures. Either anesthesia or surgical trauma plays an important role in this effect. Identification of functional lymphocyte subsets, by using appropriate monoclonal antibodies and analysis of flow cytometry data, appears to provide an accurate measurement of cellular immune competence. We found a significant decrease in the total number of T helper/inducer cells (p<0.035), B cells (p<0.043) and natural killer cells (NK) (p<0.018) but in contrast, increase in NK cell activity (p<0.012) in the peripheral arterial blood of ten patients undergoing coronary artery bypass grafting with cardiopulmonary bypass (group 1) immediately after surgery and postoperative day 1 (POD1). On the other hand, there was no significant change of these parameters occurred in the peripheral arterial blood of ten patients (group 2) who were undergoing coronary artery bypass grafting without cardiopulmonary bypass. Therefore, we conclude that coronary artery bypass grafting (CABG) with cardiopulmonary bypass induce a greater decrease in immunologic response than CABG without cardiopulmonary bypass (off pump) operations. Nevertheless, off pump CABG operations do not induce a greater decrease in immunologic response than other surgical operations.

Transforming growth factor beta-1 level in pleural effusion.

Objective:  Transforming growth factor‐β1 is an important immunomodulator. The diagnostic role of TGF‐β1 has not been systematically investigated in pleural effusion.

Flow cytometric assay of platelet glycoprotein receptor numbers in hypercholesterolemia

In this study, platelet glycoprotein (Gp) receptor numbers were measured by a flow cytometric assay using Cytoquant Gp in seven hypercholesterolemic and five normal subjects. Thrombin receptor agonist peptide (TRAP) was used to activate platelets. In hypercholesterolemia the Gp receptor numbers per resting platelet were found to be: 38 629 - 8538 (GpIIb/IIIa), 22 269 - 5628 (GpIb), 37 037 - 9810 (GpIIIa), 224 - 504 (CD62-P). After activation, receptor numbers were determined to be: 56 399 - 9003 (GpIIb/IIIa), 10 970 - 5319 (GpIb), 50 715 - 7904 (GpIIIa), 1222 - 687 (P-selectin). In the normal group before the activation, receptor numbers were: 43 828 - 8862 (GpIIb/IIIa), 22 166 - 3847 (GpIb), 42 351 - 1049 (GpIIIa), 62 - 139 (CD62-P), After activation, receptor numbers were determined to be: 60 573 - 4294 (GpIIb/IIIa), 13 003 - 4118 (GpIb), 52 067 - 1039 (GpIIIa), 3608 - 1508 (CD62-P). In hypercholesterolemic subjects, GpIIb/IIIa and GpIIIa receptor numbers on activated platelets increased significantly, whereas P-selectin numbers remained unchanged. However, the GpIb levels decreased significantly. In the control group, after activation, GpIIb/IIIa and P-selectin receptors increased significantly, GpIIIa receptor numbers did not change significantly, whereas GpIb receptor numbers decreased significantly. When the GpIIb/IIIa, GpIb, GpIIIa receptor numbers of the control group and hypercholesterolemic group were compared before and after activation, no significant changes were observed ( P > 0.05). But P-selectin receptor numbers were significantly decreased in hypercholesterolemic patients compared to normals following TRAP activation ( P < 0.05). In this study, the effect of hypercholesterolemia on platelet function was observed. The striking observation about present study was the marked decrease in P-selectin expression after activation in the hypercholesterolemics compared to normals. This finding suggests some sort of platelet dysfunction in these individuals.

Prevalence and Levels of Serum Antibodies to Gram Negative Microorganisms in Turkish Patients with HLA-B27 Positive Acute Anterior Uveitis and Controls

Purpose: Acute anterior uveitis (AAU), seronegative spondyloarthropathies such as ankylosing spondylitis and reactive arthritis form the group of “HLA-B27-associated diseases.” The aim of this study was to analyze the prevalence and levels of serum antibodies against gram negative bacteria in Turkish patients with AAU. Methods: Twenty-five patients each with previously diagnosed HLA-B27 positive and negative AAU and 25 age-and sex-matched healthy control subjects were included in the study. Serum IgM, IgG and IgA antibodies to Yersiniae enterocolitica, Campylobacter jejuni and Chlamydia trachomatis were measured using enzyme linked immunosorbent assay (ELISA). Categorical data were analyzed by chi-square test. Serum levels were analyzed using the Kruskal-Wallis test. Results: The prevalence of serum IgM, IgG and IgA antibodies did not differ significantly between the HLA-B27 positive AAU, HLA-B27 negative AAU and control groups with the exception of serum IgA antibodies against Yersiniae enterocolitica. IgA antibody against Yersiniae enterocolitica was found to be more frequently positive in the control group. Comparison of serum IgM, IgG and IgA antibody levels did not significantly differ between three groups. Conclusions: Serum antibody positivity against Yersiniae enterocolitica, Campylobacter jejuni and Chlamydia trachomatis is not frequent in the HLA-B27 positive and negative AAU patients. Serum levels of IgM, IgG, and IgA antibodies also did not show significant difference between three groups. No association between these microorganisms and the etiology of AAU was evident.

Effect of Interferon Alfa-2a on Peripheral Blood CD4+CD25+ T Regulatory Cells in Patients with Behçet Uveitis: Preliminary Study

Purpose: To evaluate the phonotypical and functional effect of interferon alfa-2a therapy on peripheral blood CD4+CD25+ T regulatory (Treg) cells in patients with Behcet uveitis. Methods: Blood was taken from 5 patients with refractory Behcetpanuveitis and 5 age-matched healthy controls. Flow cytometric analysis of CD4+CD25+Treg cells was performed. CD4+CD25+Treg cells were separated by magnetic-assisted cell sorting and co-cultured. Cytokine levels in the supernatants were determined by ELISA. Results: The percentage of CD4+CD25+Foxp3+Treg cells and the intensity of Foxp3 expression of CD4+CD25+Treg cells were slightly elevated in patients when compared to controls. Interferon alfa-2a led to a borderline significant decline of CD4+CD25+Foxp3+Treg cells and elevation of interleukin-10 (p=0.06). Conclusions: Interferon alfa-2a therapy might lead to a decline in the dysfunctional CD4+CD25+Foxp3+Treg cell population. Interleukin-10 may play a major role in IFN α-2a mediated control of Behcet uveitis.

Effects of Malnutrition on Neutrophil/Mononuclear Cell Apoptotic Functions in Children with Acute Lymphoblastic Leukemia

ABSTRACT Background: Recent studies claim that apoptosis may explain immune dysfunction observed in malnutrition. Objective: The objective of this study was to determine the effect of malnutrition on apoptotic functions of phagocytic cells in acute lymphoblastic leukemia (ALL). Materials and Methods: Twenty-eight ALL patients (13 with malnutrition) and thirty controls were enrolled. Neutrophil and mononuclear cell apoptosis of ALL patients and the control group were studied on admission before chemotherapy and repeated at a minimum of three months after induction of chemotherapy or when the nutritional status of leukemic children improved. Results: The apoptotic functions of both ALL groups on admission were significantly lower than those of the control group. The apoptotic functions were lower in ALL patients with malnutrition than those in ALL patients without malnutrition, but this was not statistically significant. The repeated apoptotic functions of both ALL groups were increased to similar values with the control group. This increase was found to be statistically significant. Conclusions: The apoptotic functions in ALL patients were not found to be affected by malnutrition. However, after dietary intervention, increased apoptotic functions in both ALL patient groups deserve mentioning. Dietary intervention should always be recommended as malnutrition or cachexia leads to multiple complications. Enhanced apoptosis might originate also from remission state of cancer.

Immune and inflammatory gene expressions are different in Behçet's disease compared to those in Familial Mediterranean Fever.

OBJECTIVE The immune classification of Behçets disease (BD) is still controversial. In this study, we aimed to compare the immune/inflammatory gene expressions in BD with those in familial Mediterranean fever (FMF), an autoinflammatory disorder with innate immune activation. MATERIAL AND METHODS CD4+ T cells and CD14+ monocytes were isolated from the peripheral blood mononuclear cells of Behçets disease patients (n=10), FMF (n=6) patients, and healthy controls (n=4) with microbeads, and then, the mRNA was isolated. The expressions of 440 genes associated with immune and inflammatory responses were studied with a focused DNA microarray using a chemiluminescent tagging system. Changes above 1.5-fold and below 0.8-fold were accepted to be significant. RESULTS In BD patients, in the CD4+ T-lymphocyte subset, interleukin 18 receptor accessory protein (1.7-fold), IL-7 receptor (1.9-fold), and prokineticin 2 (2.5-fold) were all increased compared to those in FMF patients, whereas chemokine (C-X3-C motif ) receptor-1 (CX3CR1) (0.7-fold) and endothelial cell growth factor-1 (0.6-fold) were decreased. In the CD14+ monocyte population, the V-fos FBJ murine osteosarcoma viral oncogene homolog (1.5-fold), Interleukin-8 (IL-8) (2.1-fold), and Tumor Necrosis Factor alpha (TNF-α) (1.8-fold) were all increased, whereas the chemokine (C-C motif ) ligand 5 (CCL5) (0.6-fold), C-C chemokine receptor type 7 (0.6-fold), and CX3CR1 (0.7-fold) were decreased, again when compared to those in FMF. Compared to healthy controls in the CD4+ T-lymphocyte population, in both BD and FMF patients, pro-platelet basic protein and CD27 had elevated expression. In BD and FMF patients, 24 and 19 genes, respectively, were downregulated, with 15 overlapping genes between both disorders. In the CD14+ monocytes population, chemokine (C-C motif ) receptor-1 (CCR1) was upregulated both in BD and FMF patients compared to that in the controls, whereas CCL5 was downregulated. CONCLUSION Immune and inflammatory gene expressions seem to be variable in both the innate (CD14+) and adaptive (CD4+) immune responses in BD and FMF patients compared to those in controls, suggesting differences in immune regulation between the two disorders.

Factors Affecting Stem Cell Mobilization in Patients Treated With Hematopoietic Peripheral Stem Cell Transplantation

Objectives: To assess the factors affecting stem cell mobilization in patients treated with hematopoietic peripheral stem cell transplantation. Patients and Methods: Autologous bone marrow transplants in 143 patients with 169 stem cell harvesting procedures were analysed retrospectively. Results: Stem cell mobilization was done with Filgrastim in 89 patients (52.7 %) and with Lenograstim in 80 patients (47.3%). For stem cell harvesting, Fresenius apheresis device was used in 69 patients (40.8%), while Haemonetics apheresis device was used in 100 patients (59.2%). In univariate analysis, patient’s diagnosis (p=0.005), number of treatment lines before the apheresis procedure (p=0.0004), number of leukocytes and CD34+ cell count at the first day of the apheresis procedure (p=0.0001 and p=0.0005, respectively), mobilization with filgrastim (p=0.00004) and mobilization with the Fresenius apheresis device (p=0.007) were statistically significant. In multivariate analysis, diagnosis of the patient (p=0.01), mobilization with filgrastim (p= 0.001), mobilization with Fresenius apheresis device (p=0.03), and leukocyte count at first day of apheresis (p=0.006) were important factors affecting peripheral stem cell mobilization. Conclusion: Patient’s diagnosis, mobilization with filgrastim and Fresenius apheresis device, peripheral blood leukocyte count at the first day of apheresis seem to be important in affecting peripheral stem cell mobilization.