Elżbieta Górska

Statins impair antitumor effects of rituximab by inducing conformational changes of CD20.

Background Rituximab is used in the treatment of CD20+ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas. Methods and Findings Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a 51Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells. Conclusions Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.

The role of natural killer cells in pathogenesis of autoimmune diseases

There is growing evidence that NK cell-mediated immunoregulation plays an important role in the control of autoimmunity. NK cells are a subset of lymphocytes that generally contribute to innate immunity but have also a great impact on the function of T and B lymphocytes. The major role of NK cells is cytotoxic reaction against neoplastic, infected and autoreactive cells, but they regulatory function seems to play more important role in the pathogenesis of autoimmune diseases. Numerous studies suggested the involvement of NK cells in pathogenesis of such a common autoimmune diseases as juvenile rheumatoid arthritis, type I diabetes and autoimmune thyroid diseases. The defects of NK cells regulatory function as well as cytotoxic abilities are common in patients with autoimmune diseases with serious consequences including HLH hemophagocytic lymphocytosis (HLH) and macrophage activation syndrome (MAS). The early diagnosis of NK cells defect responsible for the loss of the protective abilities is crucial for the prevention of life-threatening complications and implementation of necessary treatment.

Antiendothelial Cells Antibodies in Patients with Systemic Sclerosis in Relation to Pulmonary Hypertension and Lung Fibrosis

Although scleroderma is generally considered a fibrosing disease, it is now recognized that the underlying vascular pathology is playing a fundamental role in its pathogenesis. The present study was aimed at testing the prevalence of anti-endothelial cell antibodies (AECA) in systemic scleroderma (SSc) patients with and without pulmonary hypertension (PH) and in relation to the presence of pulmonary fibrosis. Fifty four SSc patients (50 females and 4 male, mean age 55.7 ± 16.3 years) were prospectively screened. All patients underwent transthoracic echocardiography with the estimation of pulmonary artery pressure (PAP) and tricuspid regurgitant peak gradient (TRPG). All patients suspected to have pulmonary hypertension were referred for right heart catheterization. Restrictive lung disease was confirmed by HRCT. A healthy control group included (n = 27; 7 men and 20 women, mean age 49.8 ± 12.1 years). The study of AECA was performed using the indirect immunofluorescence method on commercially available human umbilical vein endothelial cells. The HRCT scans in patients with suspected interstitial lung disease revealed signs of lung fibrosis in 15 (out of the 36 examined patients). TRPG at rest of 31 mmHg was demonstrated in 14 (21%) patients. During cardiac catheterization, arterial PH was found in two patients. Resting venous PH was found in one patient and an excessive post capillary PAP elevation at rest was demonstrated in 11 patients. At the baseline, 14/54 patients (26%) were positive for AECA. In the control group, the frequency of the antibodies was 3/27 (11%). No statistical correlation between antibody titter and the presentation of the disease existed. AECA were highly prevalent in a subgroup of patients suffering from interstitial pulmonary fibrosis. Out of the 15 patients suffering from lung fibrosis, 7 were AECA positive. The presence of AECA correlated very well with antinuclear antibodies (ANA), but was not related to the profile of ANA. Our findings support evidence that endothelial cell damage is involved in SSc, as there was increased prevalence of circulating AECA of the IgG isotype in SSc patients. AECA may also be related to the complications of SSc, like pulmonary fibrosis.

Markers of Bone Metabolism in Children with Nephrotic Syndrome Treated with Corticosteroids

The aim of the study was to assess bone mineral density, bone metabolism markers, and vitamin D level in children with idiopathic nephrotic syndrome in the course of 1-year observation. Twenty five children with nephrotic syndrome aged 5-17 years were enrolled into the study. The median number of relapses was 6 (range 1-22). All patients were treated with prednisone and vitamin D (800 IU/day). Bone mineral density of total body (TB-BMD) and lumbar spine (L-BMD), evaluated by dual energy X-ray absorptiometry (DXA) expressed as Z-score, and serum calcium, phosphorus, parathormone (iPTH), alkaline phosphatase (ALP), bone alkaline phosphatase (BAP), osteocalcin (OC), albumin, creatinine, 25(OH)D3, 1,25(OH)2D3 and urine calcium/creatinine ratio (uCa/Cr) were evaluated at the enrollment visit and after 1 year of therapy. After 1 year significant decreases of TB-BMD Z-score (from -0.24±1.34 to -0.74±1.31, p<0.05) and 25(OH)D3 serum level (from 31.7±16.3 to 23.7±9.3; p<0.05) were observed. No other appreciable differences were found. At the study onset, negative correlations were found between L-BMD Z-score and serum ALP, BAP, and phosphorus and between TB-BMD Z-score and urine uCa/Cr. After 1 year, L-BMD Z-score correlated negatively with serum BAP and OC, and positively with serum 25(OH)D3. Multivariate analysis showed that BAP was the strongest predictor of L-BMD Z-score (beta=-0.49; p<0.05). We conclude that children with nephrotic syndrome treated with corticosteroids are at risk of bone mass loss. Serum BAP concentration seems to be a good indicator of spongy bone metabolism in these children, who should be supplemented with vitamin D in an adjustable dose, possibly higher than 800 IU/24 h to prevent osteopenia.

E-selectin and sICAM-1, biomarkers of endothelial function, predict recurrence of venous thromboembolism

BACKGROUND Risk factors for atherosclerosis and venous thromboembolism (VTE) overlap and are mostly associated with endothelial dysfunction (ED). We hypothesized that ED is present in patients after the first episode of acute pulmonary embolism (APE) and predicts the risk of VTE recurrence. DESIGN AND METHODS Patients, at least 6months after the first episode of symptomatic, confirmed APE were included in this case-control study. The exclusion criteria were risk factors for cardiovascular diseases and other conditions associated with endothelial dysfunction. Eighty two patients (aged 38±11years; 44 M; 38 F) were enrolled in the study, 39 after provoked APE, 43 after unprovoked APE, and 30 controls (C) (aged 38±12years; 15 M, 15 F). In order to evaluate the endothelial function in patients with a history of APE flow-mediated dilation (FMD) of the brachial artery and biomarkers of endothelial dysfunction (sVCAM-1, sICAM-1, ADMA, E-selectin) were measured. Subsequently all patients were followed up in an outpatient clinic for VTE recurrence. RESULTS FMD was more often impaired in APE patients than in controls (5.3% (0.8-20.3) vs. 13.8% (4.1-24.3); p<0.0001). Biomarker levels differed between APE and C groups: sVCAM-1 (631ng/ml (105-2382) vs. 495ng/ml (348-934); p=0.04) and sICAM-1 (679ng/ml (279-1006) vs. 600ng/ml (394-766); p=0.002). There were 19 recurrences of VTE during the at least 12-month follow-up (15 with history of unprovoked-APE and 4 after provoked-APE). E-selectin ≥39ng/ml and sICAM-1≤655ng/ml indicated the group without recurrence of VTE. In a group of 43 unprovoked APE patients both E-selectin<39ng/ml and sICAM-1>655ng/ml were found in 17 subjects. Eleven of them (65%) were diagnosed with recurrent VTE. CONCLUSIONS Endothelial function is significantly impaired in patients after an episode of APE as indicated by FMD assessment and biomarker levels. Low concentrations of E-selectin and high levels of sICAM-1 are associated with a high risk of recurrent thromboembolism.

Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis.

Abstract Background: The eosin-5′-maleimide (EMA) binding test is a flow cytometric test widely used to detect hereditary spherocytosis (HS). EMA binds to plasma membrane proteins of red blood cells (RBCs), mainly to band 3 protein. The mean fluorescence of EMA-stained RBCs in HS patients is lower when compared with control RBCs due to the decreased amount of target proteins. EMA dye in aqueous solution is sensitive to light and high temperature. Its fluorescence can decrease when exposed to light or ambient temperatures higher than 4°C. The aim of the study was to evaluate the stability of fluorescence readings of EMA-labeled RBCs over a period of 24 h. Methods: The EMA test was performed in peripheral blood from 35 patients with microcytic anemia (five with HS, and 30 without HS). Peripheral blood samples were stained immediately after blood collection and analyzed using a flow cytometer at three time points: 0, after 1 and 24 h of storage at 4°C in the darkness. The results are presented as the percentage of normal control RBCs fluorescence. Flow cytometric studies were performed with Cytomics FC500 (Beckman Coulter, USA). Results: In HS patients the mean result of the test reached 66.72%±9.26% of normal controls, and in non-HS patients the EMA result was 99.48%±5.03% of normal control cells. The results of patients with HS were 66.72%±9.26%, 66.90%±10.24% and 67.86%±11.31% at 0 h, and after 1 and 24 h of storage, respectively. The results obtained from non-HS patients at time 0, after 1 and 24 h of storage reached 99.48%±5.03%, 99.49%±5.34% and 99.78%±6.13%, respectively. There was no difference between the results from each time point in samples from patients with or without HS. Conclusions: Results of the EMA binding test do not depend on storage time of stained samples when stored at 4°C up to 24 h after staining.

Pro- and antiangiogenic markers in patients with pulmonary complications of systemic scleroderma.

Systemic sclerosis (SSc) is an autoimmune disorder characterized by skin and internal organs fibrosis and concomitant vascular abnormalities. Although SSc is considered mainly fibrosing disease, underlying vascular pathology plays a fundamental role in its pathogenesis. We have focused on positive and negative serum markers of angiogenesis and fibrosis (pigment epithelium-derived factor [PEDF], vascular endothelial growth factor [VEGF], and soluble VEGF receptor [sVEGFR]), in progressive SSc patients at baseline and after follow-up in relation to cardiopulmonary complications (systemic hypertension [HT], pulmonary arterial hypertension [PAH] and pulmonary fibrosis [PF]). VEGF and PEDF but not sVEGFR were reciprocally regulated in SSc progression. Moreover, VEGF/PEDF ratio significantly increased during follow up suggesting that it might be used as a biomarker of disease progression. No correlation between the studied markers and cardiopulmonary complications was observed. In conclusion, VEGF and PEDF level, and the VEGF/PEDF ratio are significantly changed in the course of SSc progression and these markers can be used to assess SSc activity.

Recurrent respiratory tract infections in children - analysis of immunological examinations.

Background Paediatric respiratory tract infections are among the most common reasons for preschool and school absences and visits to physicians. The disease mainly involves the upper respiratory tract and is associated with fever, cough, sore throat, and running nose. Children with recurrent respiratory infections (RRI), which are defined as more than six serious diseases a year, are a difficult diagnostic challenge. The aim of this study was to assess immunological deviations in laboratory tests performed in children with RRI. Material and methods In the retrospective study 25 children suffering from recurrent respiratory tract infection, aged 4.1 ±2.3 years, 13 boys and 12 girls, were involved. For all children chemiluminescence of granulocytes and immunophenotyping of lymphocytes from peripheral blood were examined. An immunophenotype of peripheral blood lymphocytes involved evaluation of T cell, B cells, and NK cells, examined with flow cytometry. Results Eleven of the studied children had decreased chemiluminescent response to stimulants, normal response was found for nine children, and five children had an increased result of the test. Five of the 25 children had decreased B cells number, and five had decreased number of T cells including decrease of CD4, as well as CD8 positive cells. Children with decreased chemiluminescence had more frequent neutropaenia than children with normal or increased chemiluminescent response, p < 0.05 (exact Fisher test). Conclusions Recurrent respiratory tract infection could be associated with improper neutrophils response to pathogens, and immunological examination should be performed to find the reason for the increased number of infections in a year.

The usefulness of flow cytometric analysis of cytokines in peripheral blood and bone marrow plasma.

INTRODUCTION Recently attention has been paid to the role of cytokines in clinical pathology, since they can mediate a wide variety of biological effects. For these reasons multiplex methods have been developed to simultaneously measure numerous cytokines in individual small volume specimens. The aim of the study was to assess the usefulness of flow cytometric analysis of cytokines in peripheral blood and bone marrow plasma with Cytometric Bead Array (CBA) kits. MATERIAL AND METHODS The study involved 59 children. Tests were performed in peripheral blood and bone marrow plasma. Human Inflammatory Cytokine Kit (IL-8, -1β, -6, -10, TNF, -12p70) and Human Th₁/Th₂/Th₁₇ Cytokine Kit (IL-2, -4, -6, -10, TNF, INF-γ, -17A) (BD Bioscience) were used. Samples were analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter). RESULTS In patients diagnosed for hemophagocytic lymphohistiocytosis (HLH) (n=10) and for acute lymphoblastic leukemia (ALL) (n=12) Human Inflammatory Cytokine Kit was used. In almost all samples individual cytokines were detected in a wide range of concentrations (0.47 - 653.74 pg/ml). In samples from patients suffering from allergy (n=12) and in healthy children (n=25) Human Th₁/Th₂/Th₁₇ Cytokine Kit was used. Detection of individual cytokines was much lower: concentration range 0.09-30.17 pg/ml. DISCUSSION Based on our analysis the CBA test is suitable for analysis of several cytokines in small volumes of samples. A simple flow cytometer can be used for this test. The CBA test is more suitable for samples with expected increased levels of cytokines. When the levels of cytokines are low, the sensitivity of the CBA test can be too low.

Flow Cytometry in Detection of Abnormalities of Natural Killer Cell

The population of natural killer (NK) cells is very heterogeneous and plays a role in the immune system. Several NK cells subpopulations are recognized, differing in phenotype, cytokine release and cytotoxic ability. Different expression of biologically relevant molecules on the surface of NK cells may indicate their multiple functions. The activity of NK cells has mainly to do with their cytotoxic nature. A complete analysis of NK cells function requires application of many tests because a defect may be present at different stages of the cytotoxic process, from signal transduction through lysosome degranulation to target cells destruction. Flow cytometry is actually one of the best methods for the identification of NK cells and tracking their defects.