Milena Dąbrowska

Relationship between circulating endothelial progenitor cells and endothelial dysfunction in children with type 1 diabetes: a novel paradigm of early atherosclerosis in high-risk young patients.

OBJECTIVE The low number of circulating endothelial progenitor cells (EPCs) has emerged as a biomarker of cardiovascular (CV) risk in adults. Data regarding EPCs in paediatric populations with CV risk factors are limited. The aim of the study was to estimate the EPC number and its relationship with vascular function and structure in children with type 1 diabetes mellitus (T1DM). DESIGN AND METHODS We performed a comparative analysis of 52 children with T1DM (mean age 14.5 years; diabetes duration, 6.0 years; HbA1c level, 8.5%) and 36 healthy age- and gender-matched control children. EPCs were identified and analysed by flow cytometry with the use of MABs directed against CD34, CD144 (VE-cadherin) and CD309 (VEGFR-2). sICAM-1, hsCRP, thrombomodulin and adiponectin levels were also assessed. We evaluated vascular function (flow-mediated dilation (FMD)) and structure (carotid intima-media thickness (IMT)) ultrasonographically. RESULTS Frequencies of CD34+ cells were similar in both groups (P=0.30). In contrast, frequencies of CD34+VE-cadherin+ cells were significantly higher in diabetic children compared with the healthy group (P=0.003). Similarly, diabetic patients tended to present with higher frequencies of CD34+VEGFR+ cells (P=0.06). FMD was lower (6.9 vs 10.5%, P=0.002) and IMT was higher (0.50 vs 0.44 mm, P=0.0006) in diabetic children. We demonstrated a significant relationship between CD34+VEGFR-2+ cells and BMI (r=0.3, P=0.014), HDL (r=-0.27, P=0.04), sICAM-1 (r=0.47, P=0.023) and FMD (r=-0.45, P<0.001). Similarly, frequencies of CD34+VE-cadherin+ cells were significantly correlated with BMI (r=0.32, P=0.02) and FMD (r=-0.31, P=0.03). CONCLUSIONS We demonstrated here that increased frequencies of EPCs observed in diabetic children are negatively correlated with endothelial function. Further studies are warranted to assess whether this phenomenon might result from effective mobilisation of EPCs in order to repair damaged endothelium in children at increased risk for atherosclerosis.

Study of the protective effect of calcium channel blockers against neuronal damage induced by glutamate in cultured hippocampal neurons

BACKGROUND The aim of this study was to examine the putative protective effect of calcium channel blockers on hippocampal neurons in the experimental model of excitotoxic damage. METHODS Seven-day old primary dissociated cultures of rat hippocampal neural cells containing one of the following calcium channel blockers: cinnarizine, flunarizine or nimodipine were exposed to glutamate-induced injury. Quantitative assessments of neuronal injury were accomplished by measuring lactate dehydrogenase (LDH) activity in the media 24 h after exposure to glutamate and by counting and establishing the apoptotic and necrotic cells in flow cytometry with Annexin V-FITC/PI staining. RESULTS In our experiment, glutamate induced a 339% elevation of apoptotic cells and a 289% increase of necrotic cells in hippocampal neurons as compared to control cultures without drugs. In cultures containing flunarizine, glutamate-induced cell apoptosis was suppressed by 62% while necrosis showed no significant alternation. Cinnarizine exerted no anti-apoptotic effects on glutamate-injured cultured hippocampal neurons, while nimodipine intensified the apoptotic pathway of cell death and promoted an increase in the number of apoptotic neurons by 26%. When cinnarizine or nimodipine were used, the percentage of necrotic cells was significantly lower when compared with glutamate-injured cultures and it amounted to 44% and 24% for cinnarizine and nimodipine, respectively. CONCLUSIONS The obtained results suggest the beneficial anti-apoptotic potential of flunarizine and the anti-necrotic potential of cinnarizine against glutamate-induced death of cultured hippocampal neurons. Nimodipine can protect neurons against necrosis, but has an intensified adverse pro-apoptotic effect on cultured neurons in the experimental model of excitotoxic injury.

The expression of the chemokine receptor CCR5 in tick-borne encephalitis

BackgroundChemokine receptor 5 (CCR5) is hypothesized to drive the lymphocyte migration to central nervous system in flavivirus encephalitis, and the non-functional CCR5Δ32 genetic variant was identified as a risk factor of a West Nile virus infection and of tick-borne encephalitis (TBE). We have attempted to investigate how CCR5 expression corresponds to the clinical course and severity of TBE.MethodsWe have repeatedly studied CCR5 expression in 76 patients during encephalitic and convalescent TBE phase, analyzing its association with clinical features, cerebrospinal fluid (csf) pleocytosis, and concentrations of CCR5 ligands (chemokines CCL3, CCL4, and CCL5) and CCR5 genotype. Fifteen patients with neuroborreliosis, 7 with aseptic meningitis, 17 in whom meningitis/encephalitis had been excluded, and 18 healthy blood donors were studied as controls. Expression of CCR5 was measured cytometrically in blood and csf-activated Th lymphocytes (CD3+CD4+CD45RO+). Concentrations of chemokines in serum and csf were measured immunoenzymatically, and CCR5Δ32 was detected with sequence-specific primers. Data were analyzed with non-parametric tests, and p < 0.05 was considered significant.ResultsThe blood expression of CCR5 did neither differ between the groups nor change in the course of TBE. The CCR5 expression in the inflammatory csf was several-fold increased in comparison with blood but lower in TBE than in neuroborreliosis. The csf concentration of CCL5 was increased in TBE, the highest in the most severe presentation (meningoencephalomyelitis) and correlated with pleocytosis. The CCR5Δ32/wt genotype present in 7 TBE patients was associated with a decreased CCR5 expression, but enrichment of csf Th population in CCR5-positive cells and the intrathecal inflammatory response were preserved, without a compensatory increase of CCL5 expression.ConclusionsWe infer CCR5 and CCL5 participate in the response to TBE virus, as well as to other neurotropic pathogens. The intrathecal response to TBE is not hampered in the bearers of a single copy of CCR5Δ32 allele, suggesting that the association of CCR5Δ32 with TBE may be mediated in the periphery at the earlier stage of the infection. Otherwise, a variability of the CCR5 expression in the peripheral blood lymphocytes seems not to be associated with a variable susceptibility to TBE.

Utility of laboratory tests in B-CLL patients in different clinical stages

The study objective was to analyse the utility of laboratory tests performed in 30 patients with B-cell chronic lymphocytic leukaemia (B-CLL) at different clinical stages. Laboratory tests included automated and microscopic assessment of peripheral blood and bone marrow counts as well as evaluation of leukaemic cells. Apart from the diagnostic and prognostic value of laboratory abnormalities such as clonal lymphocytosis with CD5+CD19+CD23+ phenotype, reduced erythrocyte parameters, thrombocytopenia or bone marrow infiltration by the neoplastic clone as well as low percentage of Gumprecht’s shadows, low apoptotic activity of peripheral blood lymphocytes, and increased percentage of CD38− and ZAP-70 ± cells markedly correlate with the stage of disease progression. These results seem to confirm the diagnostic and prognostic significance of these parameters determined in routine laboratory tests in B-CLL patients.

Enhanced pretreatment CD25 expression on peripheral blood CD4+ T cell predicts shortened survival in acute myeloid leukemia patients receiving induction chemotherapy

BACKGROUND Recently, identification of CD25 (interleukin-2 receptor alpha) expression on leukemic blasts was correlated to early treatment failure and unfavorable outcome in acute myeloid leukemia (AML) patients. Here we wished to determine whether quantification of CD25 on peripheral blood CD4+ T cells could improve prognostication in newly diagnosed AML patients. METHODS The mean fluorescence intensity (MFI) of CD25 expression and frequencies of peripheral blood CD4+ T cells with varying levels of CD25 and CD127 expression were assessed by flow cytometry in all studied individuals. RESULTS Using univariate (unadjusted) and multivariate (adjusted) analyses we demonstrated that detection of high pretreatment CD25 expression on circulating CD4+ T cells was associated with significantly decreased survival rate of AML patients subjected to standard induction chemotherapy. These associations held true for both entire group of analyzed AML patients and different subgroups of patients identified by presence or absence of favorable and adverse molecular prognostic factors. CONCLUSIONS Our data indicate that quantification of CD25 expression on peripheral blood CD4+ T cells could become a novel, easily accessible method of shortened survival prognostication of AML patients subjected to standard cytotoxic therapy.

Expression of Fas receptor on human T lymphocytes under stimulation with Borrelia burgdorferi sensu lato – preliminary results

PURPOSE Apoptosis of activated T lymphocytes is essential to immunoregulation and its abnormalities have been observed in immune system disorders and persistent infections. To asses Borrelia burgdorferi influence on the susceptibility of T lymphocytes to apoptosis, we have measured expression of the Fas death receptor on these cells after incubation with live B. burgdorferi. MATERIAL AND METHODS Peripheral blood mononuclear cells from 23 LD patients (18 with Lyme arthritis, 5 with neuroborreliosis) and 13 healthy controls (C) were incubated for 48 hours with and without live B. burgdorferi spirochetes: B. afzelii, B. garinii or B. burgdorferi sensu stricto. After incubation, Fas expression on CD3+ cells was measured cytometrically with FITC-labeled monoclonal antibody. RESULTS Median fraction of Fas-expressing T lymphocytes increased under incubation with B. burgdorferi, with more cells expressing Fas after incubation with B. burgdorferi sensu stricto than with B. garinii. There was a tendency for a higher expression of Fas on T lymphocytes from LD patients then from controls, both in unstimulated and B. burgdorferi-stimulated cultures, but it did not reach a level of statistical significance. CONCLUSIONS B. burgdorferi seems to increase Fas expression on CD3+ T lymphocytes, which may render these cells more susceptible to apoptosis. This effect is stronger for B. burgdorferi s.s. than for B. garinii genospecies.

Plasma levels of MMP-7 and TIMP-1 in laboratory diagnostics and differentiation of selected histological types of epithelial ovarian cancers

BackgroundMMP-7 and TIMP-1 may play a role in the pathogenesis of cancer disease. In this study we investigated plasma levels of selected metalloproteinase and its tissue inhibitor in comparison to plasma levels of the commonly accepted tumor markers (CA 125 and HE4) in selected histological types of epithelial ovarian cancer patients as compared to control groups: patients with a benign ovarian tumor and healthy subjects. Plasma levels of MMP-7 and TIMP-1 were determined using ELISA, CA 125 and HE4 – by CMIA methods.ResultsPlasma levels of all biomarkers studied were significantly higher in ovarian cancer patients as compared to both control groups. MMP-7 demonstrated comparable to HE4 or CA125 values of diagnostic sensitivity (SE: 61%; 68%; 58%, respectively), specificity (SP: 95%; 95%; 98%, respectively), positive (PPV: 93%; 96%; 98%, respectively) and negative predictive values (NPV: 61%; 66%; 60%, respectively) in the groups tested. The combined use of the aforementioned biomarkers resulted in a further increase in diagnostic criteria and AUC, especially in the early stages of the disease.ConclusionsThese findings suggest the usefulness of combining MMP-7 with CA 125 and HE4 in the diagnosis of epithelial ovarian cancer as a new tumor marker panel.

Flow-cytometry-based evaluation of peripheral blood lymphocytes in prognostication of newly diagnosed DLBCL patients

AIMS We aimed to analyse whether quantitative assessment of peripheral blood lymphocyte CD19(+)CD20(+)CD22(+)CD79a(+) B cells, CD3(+)CD4(+)CD5(+)CD8(+) T cells and CD4(+)CD25(+++)Foxp3high Treg can improve prognostication in DLBCL patients. METHODS The absolute count of lymphocytes, B-cells, T-cells and Treg-cells as well as the percentage of apoptotic cells were assessed by means of flow cytometry in all studied subjects. RESULTS Significantly lower level of ALC and the percentage of apoptotic cells have been observed exclusively in DLBCL patients with HR. We also showed, that in comparison with LR, in HR and MR groups, there is a significant decrease in the absolute number of T-cells and Tregs. The applied treatment does no normalize the number of B-cells, Tregs and apoptotic cells only in the case of HR patients. CONCLUSIONS Lymphopenia, the decreased absolute number of T cells, Tregs, and a percentage of apoptotic cells, correlates with clinical staging in DLBCL patients. The increased number of B cells and the decreased level of Tregs and apoptotic cells after treatment might predict a poor clinical outcome in patients treated with RCHOP. Thereby, flow-cytometry-based evaluation of peripheral blood lymphocytes may be useful in prognostication of newly diagnosed DLBCL patients.

Total knee replacement induces peripheral blood lymphocytes apoptosis and it is not prevented by regional anesthesia - a randomized study

BACKGROUND Among the many changes caused by a surgical insult one of the least studied is postoperative immunosuppression. This phenomenon is an important cause of infectious complications of surgery such as surgical site infection or hospital acquired pneumonia. One of the mechanisms leading to postoperative immunosuppression is the apoptosis of immunological cells. Anesthesia during surgery is intended to minimize harmful changes and maintain perioperative homeostasis. The aim of the study was evaluation of the effect of the anesthetic technique used for total knee replacement on postoperative peripheral blood lymphocyte apoptosis. METHODS 34 patients undergoing primary total knee replacement were randomly assigned to two regional anesthetic protocols: spinal anesthesia and combined spinal-epidural anesthesia. 11 patients undergoing total knee replacement under general anesthesia served as control group. Before surgery, immediately after surgery, during first postoperative day and seven days after the surgery venous blood samples were taken and the immunological status of the patient was assessed with the use of flow cytometry, along with lymphocyte apoptosis using fluorescent microscopy. RESULTS Peripheral blood lymphocyte apoptosis was seen immediately in the postoperative period and was accompanied by a decrease of the number of T cells and B cells. There were no significant differences in the number of apoptotic lymphocytes according to the anesthetic protocol. Changes in the number of T CD3/8 cells and the number of apoptotic lymphocytes were seen on the seventh day after surgery. CONCLUSION Peripheral blood lymphocyte apoptosis is an early event in the postoperative period that lasts up to seven days and is not affected by the choice of the anesthetic technique.

Increased expression of Fas receptor and Fas ligand in the culture of the peripheral blood mononuclear cells stimulated with Borrelia burgdorferi sensu lato

Apoptosis of the lymphocytes plays an essential role in the regulation of inflammatory/immune responses and its abnormalities may contribute to a chronic infection, persistent inflammation and autoimmunity. Its role in the pathogenesis of the late Lyme borreliosis manifestations has not been studied so far. We have measured Th lymphocyte apoptosis rate, membrane expression of pro-apoptotic Fas receptor, and supernatant concentrations of selected soluble pro- and anti-apoptotic mediators in cultures of peripheral blood mononuclear cells from 16 patients with disseminated Lyme borreliosis (6 with osteoarticular symptoms, 7 with neuroborreliosis and 3 with acrodermatitis chronica atrophicans) and 8 healthy controls. The cultures stimulated for 48h with live Borrelia burgdorferi sensu stricto, B. garinii or B. afzelii spirochetes. Fraction of the apoptotic Th (CD3+CD4+) lymphocytes and expression of Fas in this cell population was measured cytometrically and concentrations of soluble Fas, soluble Fas ligand, IL-10, IL-12 and TGF-β in culture supernatant with ELISA assays. The expression of IL-10, soluble and membrane Fas and soluble Fas ligand was increased under stimulation and higher in the presence of B. burgdorferi sensu stricto than the other species. Apoptosis rate was not affected. There was no difference between Lyme borreliosis patients and controls. IL-10 concentration correlated negatively with the membrane Fas expression and apoptosis under stimulation with B. afzelii and B. garinii. Expression of Fas/FasL system is up-regulated under stimulation with B. burgdorferi, but without corresponding increase in lymphocyte apoptosis. Variable responses observed with different B. burgdorferi species may reflect differences in the pathogenesis of the infection in vivo.