Elevated Microparticles, Thrombin-antithrombin and VEGF Levels in Colorectal Cancer Patients Undergoing Chemotherapy.

Hypercoagulable state and neoangiogenesis are common phenomena associated with malignancy. Cancer patients have increased levels of circulating endothelium-derived microparticles (EMPs), which have been hypothesized to be involved in numerous pathophysiological processes. Hemostasis and angiogenesis are also activated in colorectal cancer (CRC) patients. The study aimed to investigate potential influence of chemotherapy on EMPs, thrombin anti-thrombin complex (TAT) and vascular endothelial growth factor (VEGF) levels in CRC patients undergoing chemotherapy. The study group consisted of 18 CRC patients: 8 stage III colon cancer (CC) and 10 stage IV rectal cancer (RC) patients. EMPs, TAT and VEGF levels were assessed before chemotherapy and after the third course. Results were compared with 10 healthy subjects. EMP concentration was measured by flow cytometry, while TAT and VEGF concentrations were assayed employing ELISA. Compared to the control group, CC and RC patients had significantly higher levels of tissue factor (TF)-bearing and non-TF-bearing EMPs before and after three courses of chemotherapy. VEGF concentrations in CRC patients were higher than in the control groups and increased following chemotherapy. TAT levels were elevated in CRC patients before chemotherapy compared to healthy subjects and significantly increased after the third course of chemotherapy. No significant correlation was found either between EMP and TAT levels, or between EMP concentrations and VEGF levels in the study group. CRC patients have increased EMPs, and TAT as well as VEGF levels tend to increase during chemotherapy.

Endothelial Microparticles and Vascular Endothelial Growth Factor in Patients With Head and Neck Cancer Undergoing Radiotherapy or Radiochemotherapy.

BACKGROUND: Endothelial microparticles (EMPs) released from activated or apoptotic endothelial cells may play a role in coagulation and thrombus formation. However, there is insufficient evidence regarding the impact of EMPs on angiogenesis in patients with cancer. MATERIALS AND METHODS: Sixteen patients with head and neck cancer (HNC) undergoing radiotherapy/radiochemotherapy (RT/RCT) and 10 healthy controls were studied. Serum EMPs were counted by flow cytometry, and vascular endothelial growth factor (VEGF) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean EMP level was significantly higher in patients with HNC before RT/RCT (1,601±1,479 EMP/µl) compared to the control group (782±698 EMP/µl). The number of EMPs was not notably increased after RT/RCT (1,629±769 EMP/µl). There was no significant correlation between the plasma EMP number and concentration of VEGF before (r=0.131; p=0.625), 1 day after (r=-0.042, p=0.874), nor 3 months after RT/RCT (r=0.454, p=0.076). CONCLUSION: Released EMPs may not influence promotion of neovascularization in patients with HNC.

Endothelial Microparticles and Blood Coagulation Activation in Head and Neck Cancer Patients Undergoing Radiotherapy or Radiochemotherapy.

BACKGROUND/AIM: Endothelial microparticles (EMP) are small vesicles which are released from the endothelium and contribute to blood coagulation activation in various clinical settings. The aim of this study was to examine whether EMP influence blood coagulation activation in cancer patients during radiotherapy/radiochemotherapy (RT/RCT). MATERIALS AND METHODS: Sixteen head and neck cancer (HNC) patients undergoing RT/RCT and 10 controls were examined. EMP and thrombin-antithrombin complex (TAT) were measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Tissue factor-positive EMP (TF+EMP) were defined as CD31+/CD142+/CD42b- Results: TF+EMP were significantly elevated in HNC patients before RT/RCT (T0) (1299±1154/µl), one day after RT/RCT (T1d) (1257±603/µl) and 3 months after RT/RCT (T3m) (1289±372/µl) compared to controls (688±647/µl). TF+EMP levels at T0/T1d and T0, as well as at T1d and T3m were not significantly different. TAT levels at T0 and T1d did not differ significantly but at T3m were significantly lower compared to T0 and T1d TF+EMP and TAT concentrations were not significantly correlated at T0 (r=0.058; p=0.828), T1d (r=0.373, p=0.154) and T3m (r=-0.302, p=0.204). CONCLUSION: TF+EMP may not contribute to hemostatic abnormalities in HNC patients.

Fatty acids and sphingolipids profile in the blood plasma of experienced divers in response to hyperbaric exposure.

INTRODUCTION: Hyperbaric exposure mimics air-breathing scuba diving, which is reaching enormous popularity around the world. The diver's body is subjected to a broad range of divergent effects exerted by, e.g.: an increased partial pressure of inert gases, microclotting, oxidative stress and/or production of gas bubbles. However, very little is known regarding the impact of hyperbaric exposure on plasma fatty acids content and composition, together with the body's sphingolipids profile. MATERIAL AND METHODS: The aim of this study was to investigate the contents of major fatty acids present in the plasma as well as sphingolipids, namely: sphingosine (SPH); sphingosine-1-phosphate (S1P); sphinganine (SPA); and ceramide (CER), after hyperbaric exposure corresponding to dives conducted to the depths of 30 and 60 meters of seawater. For the plasma lipids measurements, high-performance liquid chromatography together with gas-liquid chromatography were applied. RESULTS: We demonstrated that hyperbaric exposure does not affect the content and composition of plasma fatty acids of experienced divers. Similarly, the amounts of major sphingolipids fractions were not influenced, as only the content of sphingosine-1-phosphate in the plasma was significantly decreased. CONCLUSIONS: Observed lack of significant changes in plasma lipid profile after hyperbaric exposure suggests that the procedure might be considered as secure. However, decreased sphingosine-1-phosphate content in the plasma might possibly exert some adverse effects.

High-frequency (13.56-MHz) and ultrahigh-frequency (915-MHz) radio identification systems do not affect platelet activation and functions.

BACKGROUND: In radiofrequency identification (RFID) systems used in labeling of blood components, blood cells are subjected to the direct influence of electromagnetic waves throughout the storage period. The aim of this study was to prove the safety of storage of platelet concentrates (PCs) in containers labeled with RFID tags. STUDY DESIGN AND METHODS: Ten pooled PCs obtained from 12 buffy coats each suspended in additive solution were divided into three separate containers that were assigned to three groups: control, PCs labeled with ultrahigh frequency (UHF) range tags and exposed to 915-MHz radio waves, and PCs labeled with high-frequency (HF) range tags and exposed to 13.56-MHz radio waves. PCs were stored at 20 to 24°C for 7 days. In vitro tests of platelet (PLT) function were performed on the first, fifth, and seventh days of storage. RESULTS: There were no significant differences in pH; hypotonic shock resistance; surface expression of CD62P, CD42a, or CD63; release of PLT-derived microparticles; PLT aggregation; and number of PLTs between PCs stored at a constant exposure to radio waves of two different frequencies and the control group on the first, fifth, and seventh days of storage. CONCLUSION: The results of the study indicate no impact of electromagnetic radiation generated in HF and UHF RFID systems and constant contact with the tags on the quality of stored PCs.

Surface antigen expression on peripheral blood monocytes in women with gynecologic malignancies.

BACKGROUND: Of many specialized blood cells, monocytes are gaining increasing attention for their role in neoplastic disorders. The purpose of the present investigation was to determine the expression of selected peripheral blood monocyte surface antigens in cases of cervical, endometrial, and ovarian cancers. In addition, our aim was to validate the diagnostic value of two artificial coefficients recently proposed for the diagnosis of gynecologic malignancies: Neutrophil to Lymphocyte Ratio (NLR), and Multiplication of Neutrophil and Monocyte Counts (MNM). METHODS: We studied 69 white Caucasian women with histopathologic confirmation of endometrial (N = 42), cervical (N = 13), and ovarian (N = 14) cancers. Reference Group I were women suspected of cancer but histologically nullified (N = 20), and Group II were healthy blood donors (N = 23). Expression of CD11a, CD11b, CD11c, CD16, CD54 (ICAM-1), CD62 L (L-selectin), CD64, and HLA-DR was measured with immunofluorescence in a flow cytometer. RESULTS: CD54 expression increased by ≥35.6% (p < 0.001) whilst HLA-DR decreased by ≥10.8% (p < 0.001) in all cancer subgroups and Group I as compared to blood donors. A correlation (p < 0.05) between CD54 and CD62 L was stronger in all cancers studied than in healthy subjects. There was no difference in the NLR values between any of these subgroups. Moreover, we observed an increase in MNM parameter in cases of cervical and endometrial cancer and in the Reference Group I. CONCLUSIONS: In the studied gynecologic malignancies, CD54 expression on peripheral blood monocytes is enhanced, indicating a higher transmigrational potential present in such patients, and HLA-DR expression diminished, indicating a decreased readiness of the immune system to recognize foreign antigens. The more pronounced correlation for the expression of CD54 and CD62 L in cancer suggests that monocytes uptake from the bloodstream and their local adhesion increase the pool of tumor-associated macrophages. This study challenged the suggested credibility and usefulness of the artificial parameters of MNM and NLR for the differential diagnosis of gynecologic malignancies.

Epstein-Barr virus effect on frequency of functionally distinct T cell subsets in children with infectious mononucleosis.

PURPOSE: Epstein-Barr virus is a common human pathogen which infects the great majority of population worldwide. A striking proliferation of CD8⁺ T cells is an immune response to EBV invasion of B lymphocytes during infectious mononucleosis. The aim of the study was to analyze frequencies of CD28⁺CD95⁻, CD28⁺CD95⁺, CD28⁻CD95⁺ T cell subsets putative naïve (T(N)), central (T(CM)) and effector memory (T(EM)) T cells in children with infectious mononucleosis. MATERIAL/METHODS: Multiparameter flow cytometric analysis of CD4⁺ and CD8⁺ T cell subsets was performed in 19 children with acute infectious mononucleosis. RESULTS: The CD4⁺/CD8⁺ ratio was found to be decreased (0.53) in children with infectious mononucleosis. Median T(N), T(CM), T(EM) frequencies were estimated to be 3.7, 4.5, 15.1% of CD8⁺ and 23, 59.3, 5.5% of CD4⁺ T cells, respectively. In the present study we demonstrated negative correlations between CD8⁺CD28⁺CD95⁺ and CD8⁺CD28⁻CD95⁺ T cells and both VCA IgM antibody titers and disease duration. However, no such correlation was found when subset of CD4⁺ T cells or CD8⁺CD28⁺CD95⁻ cells was compared. CONCLUSIONS: We conclude that there is a rapid decrease in the number of memory CD8⁺ T cells in early acute stage of infectious mononucleosis.

Combination of LC-MS- and GC-MS-based metabolomics to study the effect of ozonated autohemotherapy on human blood.

Ozonated autohemotherapy (O3-AHT) is a medical approach during which blood obtained from the patient is ozonated and injected back into the body. Despite an increasing number of evidence that O3-AHT is safe, this type of therapy remains controversial. To extend knowledge about the changes in blood evoked by O3-AHT, LC-MS- and GC-MS-based metabolic fingerprinting was used to compare plasma samples obtained from blood before and after the treatment with potentially therapeutic concentrations of ozone. The procedure was performed in PVC bags utilized for blood storage to study also possible interactions between ozone and plastic. By use of GC-MS, an increase in lactic acid and pyruvic acid was observed, which indicated an increased rate of glycolysis. With LC-MS, changes in plasma antioxidants were observed. Moreover, concentrations of lipid oxidation products (LOP) and lysophospholipids were increased after ozone treatment. This is the first report of increased LOPs metabolites after ozonation of blood. Seven metabolites detected by LC-QTOF-MS only in ozonated samples could be considered as novel biomarkers of oxidative stress. Several plasticizers have been detected by both techniques in blood stored in PVC bags. PVC is known to be an ozone resistant material, but ozonation of blood in PVC bags stimulates leaching of plasticizers into the blood.

[Quality of buffy-coat-derived platelet concentrates prepared using automated system terumo automated centrifuge and separator integration (TACSI)]. / Ocena jakosci koncentratów plytek krwi otrzymanych z utyciem automatycznego systemu Terumo automated centrifuge and separator integration (TACSI).

UNLABELLED: Platelet recovery, and viability, and function is strongly dependent on the method of the preparation of platelet concentrate (PC). The glucose consumption, decrease of pH, release of alpha granules during storage in platelet concentrate impair their clinical effectiveness. THE AIM OF STUDY: To compare of the quality of buffy-coat-derieved platelet concentrates prepared using automatic system terumo automated centrifuge and separator integration (TACSI) and stored over 7 days. MATERIAL AND METHODS: PCs were prepared from buffy coats using manual method (group I), or automatic system TACSI (group II). Fifteen PCs prepared from the 5 buffy coats each were stored over 7 days in 22-24 degrees C and tested. Samples were taken from the PCs container on days 1 and 7. The following laboratory tests were performed: number of platelets, platelets derived microparticles, CD62P expression, platelet adhesion, pH, glucose, lactate dehydrogenase activity. RESULTS: We have observed higher expression of CD62P in PCs prepared using manual method compared to the PCs produced automatically Platelet recovery was significantly higher in PCs prepared using automatic systems compare to manual method. CONCLUSION: Compared to manual methods, automatic system for preparation of buffy coats, is more efficient and enable production of platelets concentrates of higher quality.

[The quality of platelet concentrates in dependence on storage time before pathogen inactivation]. / Jakosc koncentratów plytek krwi w zaleznosci od czasu ich przechowywania przed wykonaniem procedury inaktywacji patogenów.

UNLABELLED: Pathogen inactivation procedure performed just before distribution of platelet concentrates (PCs) may decrease costs caused by loss of these components due to relatively short expiry date. THE AIM OF STUDY: To evaluate the quality of PCs pathogen inactivated on the first or the fifth day of storage. MATERIAL AND METHODS: PCs preparated from buffy-coats were suspended in platelet additive solution (Intersol, Baxter Healthcare Corporation, Belgium). The photochemical pathogen inactivation was performed on the 1st or the 5th day of storage using amotosalen and UVA (Cerus, Europe BV). PCs were stored for 7 days. RESULTS: There were observed increased expression of CD62 and CD63, elevated activity of LDH and lower concentration of glucose in PCs pathogen inactivated on day 1 compare to the control group. PCs pathogen inactivated on day 5 showed decreased expression of CD62 and CD63 compare to the control group. There were no significant differences in platelet number, pH, lactate concentration, hypotonic shock response and release of platelet derived microparticles in both groups of pathogen inactivated PCs. CONCLUSIONS: Time of storage of PCs before pathogen inactivation has no significant impact on PCs quality. Pathogen inactivation procedure performed just after having received request for PCs is more cost effective than the routine pathogen inactivation in all PCs before storage.

The inflammatory reaction during chronic venous disease of lower limbs.

Chronic venous disease (CVD) is an insufficiency of distal veins caused by their partial or total obstruction, endothelial distension and functional disorders. Chronic venous disease of lower limbs is common problem and affects millions of people. In this article we suggest that inflammatory process is involved in the structural remodeling in venous valves and in the venous wall, leading to valvular incompetence and the development of varicose veins.

Evaluation of the memory CD4+ and CD8+ T cells homeostasis during chronic venous disease of lower limbs.

More and more is known about the role of venous wall abnormalities and valvular incompetence in the development of chronic venous disorders (CVD). Unfortunately detailed mechanisms of CVD pathophysiology are not well understood. Recent studies focus on involvement of the inflammatory process in the structural remodeling of venous valves and venous wall. The aim of this study is to investigate and to document the memory T cells homeostasis in CVD patients. In this study we present lymphocytic changes in blood from varicose veins in terms of total CD4+ and CD8+ T cells and their particular subsets of memory T cells: TN, TCM and TEM. Results suggest that immunological memory may be involved in the CVD development.

Effector and memory CD4+ and CD8+ T cells in the chronic infection process.

T cell memory in comparison with B cell memory is not well understood. This review focuses on CD8+ and CD4+ memory T cells. In this article we try to define memory cells and also present models of memory T cells formation. We would also like to delineate their differentiation into distinct subsets. Long-lived memory T cells consist in two main subsets: TCM and TEM. Recent studies have shown that not all cells considered to be memory cells differentiate into TCM and TEM, but a small proportion of theses cells exhibit naive cells phenotype. Memory T cells constitute a heterogeneous population of cells. In this study we lay stress on characteristic of main memory T cells subsets and their alleged participation in immune response upon reexposure to the Ag.

Platelet count, mean platelet volume and thrombocytopoietic indices in healthy women and men.

UNLABELLED: Gender-dependent differences in platelet count have been demonstrated in few studies. In women platelet count is higher than in men, which seems to reflect different hormonal profiles or a compensatory mechanism associated with menstrual blood loss. The aim of the study was to assess platelet count, mean platelet volume and thrombocytopoietic indices in women and men. The study was conducted on healthy blood donors divided into groups: F - 60 women and M - 65 men. Platelet count and mean platelet volume were determined on a haematological analyser Advia 120, Bayer. The following thrombocytopoietic indices were measured: thrombopoietin concentration (ELISA), percentage of reticulated platelets (flow cytometry, COULTER EPICS XL) and absolute reticulated platelet count. RESULTS: Higher platelet count was noted in the group of women 252.35 +/- 41.25 x 10(9)/l as compared to men 221.87 +/- 37.63 x 10(9)/l (p = 0.0002). At the same time women had lower thrombopoietin concentration 156.50 +/- 57.18 pg/ml compared to men 180.46 +/- 60.98 pg/ml, (p = 0.03). No statistically significant differences were found in the mean platelet volume, percentage of reticulated platelets or absolute reticulated platelet count between group F and M. CONCLUSIONS: Platelet count is gender-dependent, being higher in women than in men. Thrombopoietin concentration is gender-dependent and is lower in women than in men. In physiological conditions, there is no correlation between platelet count and thrombopoietin concentration in women (r = -0.155) and men (r = -0.2586).

Evaluation of selected peripheral blood leukocyte functions in patients with various forms of periodontal disease.

INTRODUCTION: Periodontitis (P) is an infectious disease that develops in the supporting tissues of the tooth. One of the risk factors leading to it may be dysfunction of some immune system cells. Therefore, the object of the study was to assess selected functions of peripheral blood leukocytes in patients with various forms of P. As leukocytes are able to secrete interleukin (IL)-4 and IL-6, concentrations of their soluble receptors and the expression of their membrane receptors were investigated. MATERIAL AND METHODS: Twenty generally healthy subjects with aggressive (AP)and chronic periodontitis (CP)were enrolled in the study. The control group consisted of 8 healthy subjects,with no changes in periodontium. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured. Levels of IL-4,IL-6,and their soluble receptors sIL-4R and sIL-6R were determined in the supernatant by ELISA. The expressions of cell surface IL-4R and IL-6R were assayed on PBMC using flow cytometry. RESULTS: No statistically significant differences were found in the selected parameters between people with periodontal disease and healthy controls. However, in subjects with AP, there was an increasing tendency in IL-6 concentration and IL-4R expression on PBMCs. CONCLUSIONS: Our results show that leukocytes play a significant part in P and their activity is probably lesion-dependent. Estimation of the cytokines secreted by leukocytes may facilitate differentiation and prognosis of the disease progression.

Naive and memory T cells in hypertrophied adenoids in children according to age.

The anatomic location of the adenoid implies that this organ is the first site of contact with inhaled antigens. Depending on the expression of different isoforms of the CD45 molecules, T cells can be divided into naive (CD45RA(+)) and memory (CD45R0(+)) cells, the latter representing T cells that have already been exposed to antigens. The purpose of this study was to analyse the lymphoid cells' subsets in adenoids and relate the findings to the age. The analysed material was adenoid tissue removed on the grounds of hypertrophy from 22 children. The patients were divided into two groups: up to 5 and above 5 years of age. The analyses of the lymphocytes subpopulations in the adenoid were performed in an EPICX XL (Coulter) flow cytometry. The results are expressed as the percentage of positively labeled cells (CD4(+), CD8(+), CD4(+)/CDB(+), CD4(+)CD45RA(+), CD8(+)CD45RA(+), CD4(+)CD45R0(+), CD8(+)CD45R0(+)). The percentage of CD4(+)/CD45R0(+) in children up to 5 years of age was significantly lower than in older children. We found the positive regression between age and the percentage of CD4(+) cells was CD45R0(+) (r=0.64). There were no statistically significant differences between study subgroups for the other parameters. The positive regression for CD4(+)/CD45R0(+) cells and age may result from increased stimulation by bacterial, viral and other antigens. Our results indicate that the adenoid have an important role in the development of an immunological memory among younger children.

Lymphocyte subpopulations in hypertrophied adenoid in children with otitis media with effusion.

Otitis media with effusion (OME) usually coexist with the adenoid hypertrophy. The aim of this study was a quantitative evaluation of the lymphocyte subpopulations in hypertrophied adenoid in children with persistent (ove three months) otitis media with effusion. We used flow cytometry for study the subpopulations of the lymphocytes in the pharyngeal adenoid during otitis media in the children, alternative group consist of children with simple adenoid hypertrophy. The monoclonal antibodies selected were specific markers for T lymphocytes (CD3), T-helper cells (CD4), T-suppressor cells (CD8), B cells (CD19) and NK cells (CD56). There were also analyzed T activation markers: HLA-DR and CD25. The percentage of NK cells was significantly decreased in the group of younger children with otitis media with effusion (0.81 +/- 0.51) in comparison to older ones (1.40 +/- 0.59%). Also, in the group of younger children with adenoid hypertrophy, the NK cells percentage was significantly lower (0.85 +/- 0.37%) than in the group of older children--(1.46 +/- 0.83). In both subgroups of younger children the percentage of lymphocytes T with HLA-DR activation marker was close, but it was significantly lower in the group of older children with otitis media with effusion (4.28 +/- 3.40%) in comparison to the group of older children with adenoid hypertrophy (5.03 +/- 3.00%).