Rotational Thromboelastometry (ROTEM®) in Relation to Inflammatory Biomarkers and Clinical Outcome in COVID-19 Patients.

Background: The pathogenesis of hypercoagulability in COVID-19 patients is complex and not fully understood. Rotational thromboelastometry (ROTEM®) is a viscoelastic method that allows the definition of a patient's hemostatic profile. This study aimed to assess the relationship between ROTEM® parameters, the profile of inflammatory cytokines, and clinical outcomes in COVID-19 patients. Methods: A total of 63 participants (n = 29 symptomatic non-ICU COVID-19 patients, and n = 34 healthy controls) were prospectively included in the study. We assessed the relationship between the parameters of three ROTEM® tests (NATEM®, EXTEM®, and FIBTEM®) and levels of CRP, interleukin-8, interleukin-1ß, interleukin-6, interleukin-10, tumor necrosis factor, interleukin 12p70, and clinical outcomes. Results: ROTEM® indicated hypercoagulability in COVID-19 patients in all the tests performed. The levels of all inflammatory cytokines were significantly higher in COVID-19 patients. NATEM more frequently detected hypercoagulability in COVID-19 patients compared to EXTEM. The strongest correlations with inflammatory biomarkers and CT severity score were with FIBTEM parameters. The elevated maximum clot elasticity (MCE) in FIBTEM was the strongest predictor of poor outcomes. Conclusions: Increased FIBTEM MCE may be associated with greater severity of COVID-19. Non-activated ROTEM (NATEM test) seems to be more valuable for detecting hypercoagulability in COVID-19 patients compared to the tissue factor activated test (EXTEM).

Crosstalk of Immune Cells and Platelets in an Ovarian Cancer Microenvironment and Their Prognostic Significance.

Ovarian cancer (OC) is one of the deadliest gynecological cancers, largely due to the fast development of metastasis and drug resistance. The immune system is a critical component of the OC tumor microenvironment (TME) and immune cells such as T cells, NK cells, and dendritic cells (DC) play a key role in anti-tumor immunity. However, OC tumor cells are well known for evading immune surveillance by modulating the immune response through various mechanisms. Recruiting immune-suppressive cells such as regulatory T cells (Treg cells), macrophages, or myeloid-derived suppressor cells (MDSC) inhibit the anti-tumor immune response and promote the development and progression of OC. Platelets are also involved in immune evasion by interaction with tumor cells or through the secretion of a variety of growth factors and cytokines to promote tumor growth and angiogenesis. In this review, we discuss the role and contribution of immune cells and platelets in TME. Furthermore, we discuss their potential prognostic significance to help in the early detection of OC and to predict disease outcome.

Knee Osteochondral Defect Reconstruction With Autologous Bone Grafting and Mesenchymal Cell Transplantation.

Osteochondral defects of the knee are common in orthopaedic patients. They are challenging to treat, especially in young, highly demanding patients who do not qualify for arthroplasty. Among the many possibilities to treat osteochondral lesions presented so far, none is ideal. Because of the poor healing potential of cartilage, treatment outcomes significantly worsen with larger lesions. The treatment of large defects usually requires expensive solutions, sometimes including second-stage surgery. Using mesenchymal stem cell transplantation and cancellous bone autografts, the technique presented here for osteochondral lesion reconstruction can be effectively used to treat large osteochondral lesions in a single-stage procedure.

Salivary gland dysfunction and salivary redox imbalance in patients with Alzheimer's disease.

Alzheimer's disease (AD) is associated with the deposition of ß-amyloid in the brain. AD accounts for over 50% of cases of dementia which results from disturbances in redox homeostasis. Indeed, increased intensity of protein oxidation and nitration as well as lipid peroxidation is observed in brain areas with considerable amounts of amyloid plaques and neurofibrillary tangles. However, little is known about the oxidoreductive balance of salivary glands in AD patients. Therefore, the aim of this study was to evaluate the antioxidant barrier and oxidative/nitrosative stress biomarkers in stimulated saliva and blood of AD patients. The study was participated by 25 AD patients and 25 non-demented controls without neurological diseases or cognitive impairment, matched by age and gender to the study group. The number of patients was determined based on a previous pilot study (test power = 0.9). We found a significant decrease in the activity of erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx), increased activity of catalase (CAT) and reduced concentration of plasma non-enzymatic antioxidants (uric acid, UA and reduced glutathione, GSH). In contrast, in the stimulated saliva of AD patients we observed significantly decreased activity of all antioxidant enzymes (SOD, CAT and GPx) as well as concentration of GSH compared to the control group. The content of lipid (malondialdehyde, MDA) and protein (advanced oxidation protein products, AOPP; advanced glycation end-products, AGE) oxidation products as well as biomarkers of nitrosative stress (peroxynitrite, nitrotyrosine) was significantly higher in both saliva and plasma of AD patients compared to the controls. In AD patients, we also observed a considerable decrease in stimulated saliva secretion and salivary total protein content, and an increase in salivary ß-amyloid concentration. In conclusion, AD results in redox imbalance towards oxidative reactions, both at the level of the oral cavity and the entire body. General redox balance disturbances do not coincide with salivary redox balance disturbances. Reduction in stimulated saliva secretion in AD patients reflects secretory dysfunction of the parotid glands.

Sex-dependent dysregulation of human neutrophil responses by bisphenol A.

BACKGROUND: In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17ß-estradiol (E2). METHODS: Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park's method with latex beads and Park's test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. RESULTS: The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. CONCLUSIONS: The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

Effect of bisphenol A on human neutrophils immunophenotype.

Neutrophils (PMN) play a key role in eliciting congenital immune response. These cells are equipped with specific receptors that are located on the surface of their cell membrane. These receptors produce various signals which in turn help in the effective functioning of PMN. The activity of these cells may be modified by factors of endo- and exogenous origin, including xenoestrogens such as bisphenol A (BPA). The aim of this study was to evaluate the effect of BPA on the expression of CD11c, CD14, CD15, CD16, CD62L and CD284 compounds on the surface of neutrophils in women and men. The study material included PMN isolated from the whole blood. The cells were incubated in the presence of BPA and/or LPS. Flow cytometry technique was used to evaluate the expression of CD antigens. Studies of these receptors indicate that BPA, at a concentration corresponding to the serum level of this compound in healthy subjects as well as at higher doses, induces changes in the immunophenotype of PMN, which may lead to immunity disorders associated with the dysfunction of these cells. Moreover, the observed effects of xenoestrogen on the expression of CD11c, CD14, CD15, CD16, CD62L and CD284 differentiation markers on these cells are sex-independent.

The Inhibitory Effect of Protamine on Platelets is Attenuated by Heparin without Inducing Thrombocytopenia in Rodents.

Protamine sulfate (PS) is a polycationic protein drug obtained from the sperm of fish, and is used to reverse the anticoagulant effect of unfractionated heparin (UFH). However, the interactions between PS, UFH, and platelets are still not clear. We measured the platelet numbers and collagen-induced aggregation, P-selectin, platelet factor 4, ß-thromboglobulin, prostacyclin metabolite, D-dimers, activated partial thromboplastin time, prothrombin time, anti-factor Xa, fibrinogen, thrombus weight and megakaryocytopoiesis in blood collected from mice and rats in different time points.. All of the groups were treated intravenously with vehicle, UFH, PS, or UFH with PS. We found a short-term antiplatelet activity of PS in mice and rats, and long-term platelet-independent antithrombotic activity in rats with electrically-induced thrombosis. The antiplatelet and antithrombotic potential of PS may contribute to bleeding risk in PS-overdosed patients. The inhibitory effect of PS on the platelets was attenuated by UFH without inducing thrombocytopenia. Treatment with UFH and PS did not affect the formation, number, or activation of platelets, or the thrombosis development in rodents.

The relationships among monocyte subsets, miRNAs and inflammatory cytokines in patients with acute myocardial infarction.

BACKGROUND: Acute myocardial infarction (AMI) causes irreversible myocardial damage and release of inflammatory mediators, including cytokines, chemokines and miRNAs. We aimed to investigate changes in the levels of cytokines (IL-6, TNF-α and IL-10), miRNAs profiles (miR-146 and miR-155) and distribution of different monocyte subsets (CD14++CD16-, CD14++CD16+, CD14+CD16++) in the acute and post-healing phases of AMI. METHODS: In eighteen consecutive AMI patients (mean age 56.78 ± 12.4 years, mean left ventricle ejection fraction - LVEF: 41.9 ± 9.8%), treated invasively, monocyte subsets frequencies were evaluated (flow cytometry), cytokine concentrations were analyzed (ELISA) as well as plasma miRNAs were isolated twice - on admission and after 19.2 ± 5.9 weeks of follow-up. Measurements were also performed among healthy volunteers. RESULTS: AMI patients presented significantly decreased frequencies of classical cells in comparison to healthy controls (median 71.22% [IQR: 64.4-79.04] vs. 84.35% [IQR: 81.2-86.7], p = 0.001) and higher percent of both intermediate and non-classical cells, yet without statistical significance (median 6.54% [IQR: 5.14-16.64] vs. 5.87% [IQR: 4.48-8.6], p = 0.37 and median 5.99% [IQR: 3.39-11.5] vs. 5.26% [IQR: 3.62-6.2], p = 0.42, respectively). In AMI patients both, analyzed plasma miRNA concentrations were higher than in healthy subjects (miR-146: median 5.48 [IQR: 2.4-11.27] vs. 1.84 [IQR: 0.87-2.53], p = 0.003; miR-155: median 25.35 [IQR: 8.17-43.15] vs. 8.4 [IQR: 0.08-16.9], p = 0.027, respectively), and returned back to the values found in the control group in follow-up. miR-155/miR-146 ratio correlated with the frequencies of classical monocytes (r=0.6, p = 0.01) and miR-155 correlated positively with the concentration of inflammatory cytokines - IL-6 and TNF-α. CONCLUSIONS: These results may suggest cooperation of both pro-inflammatory and anti-inflammatory signals in AMI in order to promote appropriate healing of the infarcted myocardium.

Very Small Embryonic-Like Stem Cells, Endothelial Progenitor Cells, and Different Monocyte Subsets Are Effectively Mobilized in Acute Lymphoblastic Leukemia Patients after G-CSF Treatment.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a malignant disease of lymphoid progenitor cells. ALL chemotherapy is associated with numerous side effects including neutropenia that is routinely prevented by the administration of growth factors such as granulocyte colony-stimulating factor (G-CSF). To date, the effects of G-CSF treatment on the level of mobilization of different stem and progenitor cells in ALL patients subjected to clinically effective chemotherapy have not been fully elucidated. Therefore, in this study we aimed to assess the effect of administration of G-CSF to ALL patients on mobilization of other than hematopoietic stem cell (HSCs) subsets, namely, very small embryonic-like stem cells (VSELs), endothelial progenitor cells (EPCs), and different monocyte subsets. METHODS: We used multicolor flow cytometry to quantitate numbers of CD34+ cells, hematopoietic stem cells (HSCs), VSELs, EPCs, and different monocyte subsets in the peripheral blood of ALL patients and normal age-matched blood donors. RESULTS: We showed that ALL patients following chemotherapy, when compared to healthy donors, presented with significantly lower numbers of CD34+ cells, HSCs, VSELs, and CD14+ monocytes, but not EPCs. Moreover, we found that G-CSF administration induced effective mobilization of all the abovementioned progenitor and stem cell subsets with high regenerative and proangiogenic potential. CONCLUSION: These findings contribute to better understanding the beneficial clinical effect of G-CSF administration in ALL patients following successful chemotherapy.

Simultaneous use of erythropoietin and LFM-A13 as a new therapeutic approach for colorectal cancer.

BACKGROUND AND PURPOSE: Bruton's tyrosine kinase (Btk) is a non-receptor tyrosine kinase involved in the activation of signalling pathways responsible for cell maturation and viability. Btk has previously been reported to be overexpressed in colon cancers. This kind of cancer is often accompanied by anaemia, which is treated with an erythropoietin supplement. The goal of the present study was to assess the effects of combination therapy with erythropoietin ß (Epo) and LFM-A13 (Btk inhibitor) on colon cancer in in vitro and in vivo models. EXPERIMENTAL APPROACH: DLD-1 and HT-29 human colon adenocarcinoma cells were cultured with Epo and LFM-A13. Cell number and viability, and mRNA and protein levels of Epo receptors, Btk and Akt were assessed. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM-A13. KEY RESULTS: The combination of Epo and LFM-A13 mostly exerted a synergistic inhibitory effect on colon cancer cell growth. The therapeutic scheme used effectively killed the cancer cells and attenuated the Btk signalling pathways. Epo + LFM-A13 also prevented the normal process of microtubule assembly during mitosis by down-regulating the expression of Polo-like kinase 1. The combination of Epo and LFM-A13 significantly reduced the growth rate of tumour cells, while it showed high safety profile, inducing no nephrotoxicity, hepatotoxicity or changes in the haematological parameters. CONCLUSION AND IMPLICATIONS: Epo significantly enhances the antitumour activity of LFM-A13, indicating that a combination of Epo and LFM-A13 has potential as an effective therapeutic approach for patients with colorectal cancer.

Protective effect of cigarette smoke on the course of dextran sulfate sodium-induced colitis is accompanied by lymphocyte subpopulation changes in the blood and colon.

BACKGROUND: Cigarette smoke (CS) exerts protective effect against ulcerative colitis. The mechanism of this phenomenon remains unknown. One of the possible explanation by which CS exerts its anti-inflammatory action is modulation of immune system. Therefore, the aim of the study was to evaluate the effect of CS on the course of inflammation and subpopulations of lymphocytes in the blood and colon in mice with dextran sulfate sodium (DSS)-induced colitis. METHODS: C57BL6/cmdb mice were exposed to CS for 4 weeks. Colitis was induced with 3.5% DSS given for 10 days. Severity of colitis was determined by disease activity index (DAI), body weight changes, and macro- and microscopic characteristics of inflammation. Peripheral subpopulations of lymphocytes were assessed by flow cytometry (blood) or immunohistochemistry (colonic tissue). RESULTS: Mice treated with 3.5% DSS developed severe colitis with significantly decreased body weight, increased DAI, and macroscopic and histological features of colonic inflammation. These findings were diminished after concomitant exposure to CS. Mice exposed to DSS alone demonstrated significantly decreased percentage of total CD4+ cells (73.1 vs. 52%, p = 0.0007), accompanied by increase of CD8+ cells (18.4 vs. 39.5%, p = 0.0001). Concomitant CS exposure reversed inappropriate CD4+/CD8+ ratio both in the blood and colon and significantly increased B cell presence in the colon. CONCLUSIONS: Our study has demonstrated that CS exposure decreases severity of DSS-induced colitis. This phenomenon was accompanied by changes in CD4/CD8 ratio and B cell level in the peripheral blood and colon. These mechanisms may be responsible for protective effect of smoking in ulcerative colitis.

Involvement of hyperglycemia in the development of platelet procoagulant response: the role of aldose reductase and platelet swelling.

: Rise in mean platelet volume (MPV) has been demonstrated to be associated with increased platelet reactivity. In diabetes patients, augmented MPV was proposed to contribute to increased risk of thrombotic complications. Therefore, the aim of this study was to investigate whether under hyperglycemic conditions, aldose reductase (AR)-mediated sorbitol formation and associated rise in cell volume, which subsequently results in platelet hyperactivation. Platelets were obtained from 30 healthy volunteers and 13 patients with diabetes. We evaluated changes in platelet size, their reactivity (measured as aggregation and secretion), and sorbitol content evoked by glucose. Measurement of procoagulant activity and thromboelastography were performed to assess how hyperglycemia affects coagulation. We have found that incubation of platelets with glucose (>10 mmol/l) leads to increased MPV, potentiation of collagen-evoked platelet aggregation, secretion, and procoagulant response (measured as platelet-dependent thrombin generation and phosphatidylserine expression). Glucose-treated platelets had higher sorbitol content and demonstrated enhanced tubulin polymerization. All the above-mentioned phenomena were reduced following the blocking of AR or by vincristine (microtubule destabilizing agent). Thromboelastography measurements demonstrated that hyperglycemia is associated with reduction of clotting time (R) and increase in the alpha angle (reflects platelet activation). Addition of sorbinil (AR inhibitor) or vincristine normalized R variable and alpha angle. The hyperglycemic conditions may accelerate platelet-related thrombin generation through the activation of polyol pathway, enhanced tubulin polymerization and associated with it rise in platelet volume.

Unfavorable effect of levetiracetam on cultured hippocampal neurons after hyperthermic injury.

BACKGROUND: The aim of this study was to examine the viability of neurons and the putative neuroprotective effects of second-generation antiepileptic drug, levetiracetam (LEV), on cultured hippocampal neurons injured by hyperthermia. METHODS: Primary cultures of rat's hippocampal neurons at 7day in vitro (DIV) were incubated in the presence or absence of LEV in varied concentrations under hyperthermic conditions. Cultures were heated in a temperature of 40°C for 24h or in a temperature of 41°C for 6h. Flow cytometry with Annexin V/PI staining as well as fluorescent microscopy assay were used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, the release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. Assessment was performed after 9DIV and 10 DIV. RESULTS: Incubation of hippocampal cultures in hyperthermic conditions resulted in statistically significant increase in the number of injured neurons when compared with non-heated control cultures. Intensity of neuronal destruction was dependent on temperature-value. When incubation temperature 40°C was used, over 80% of the population of neurons remained viable after 10 DIV. Under higher temperature 41°C, only less than 60% of neurons were viable after 10 DIV. Both apoptotic and necrotic pathways of neuronal death induced by hyperthermia were confirmed by Annexin V/PI staining. CONCLUSIONS: LEV showed no neuroprotective effects in the current model of hyperthermia in vitro. Moreover, drug, especially when used in higher concentrations, exerted unfavorable intensification of aponecrosis of cultured hippocampal neurons.

The effect of penicillin administration in early life on murine gut microbiota and blood lymphocyte subsets.

BACKGROUND AND AIM: Antibiotics have many beneficial effects but their uncontrolled use may lead to increased risk of serious diseases in the future. Our hypothesis is that an early antibiotic exposition may affect immune system by altering gut microbiota. Therefore, the aim of the study was to determine the effect of penicillin treatment on gut microorganisms and immune system of mice. METHODS: 21-days old C57BL6/J/cmdb male mice were treated with low-dose of penicillin (study group) or water only (control group) for 4 weeks. Tissue and stool samples for histology or microbiome assessment and peripheral blood for CBC and flow cytometry evaluation were collected. RESULTS: We found high variability in microbiota composition at different taxonomic levels between littermate mice kept in the same conditions, independently of treatment regimen. Interestingly, low-dose of penicillin caused significant increase of Parabacteroides goldsteinii in stool and in colon tissue in comparison to control group (9.5% vs. 4.9%, p = 0.008 and 10.7% vs. 6.1%, p = 0.008, respectively). Moreover, mice treated with penicillin demonstrated significantly elevated percentage of B cells (median 10.5% vs 8.0%, p = 0.01) and decrease in the percentage of total CD4+ cell (median 75.4% vs 82.5%, p = 0.0039) with subsequent changes among subsets - increased percentage of regulatory T cells (Treg), T helper 1 (Th1) and T helper 2 (Th2) cells. CONCLUSION: Our study showed significant effect of penicillin on B and T cells in peripheral blood of young mice. This effect may be mediated through changes in gut microbiota represented by the expansion of Parabacteroides goldsteinii.

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 / Artroplastia total do joelho induz apoptose em linfócitos de sangue periférico e não é evitada por anestesia regional - Estudo randomizado

ABSTRACT 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.

RESUMO JUSTIFICATIVA E OBJETIVO: Dentre as muitas alterações causadas por uma ferida cirúrgica, uma das menos estudadas é a imunossupressão pós-operatória. Esse fenômeno é uma causa importante das complicações infecciosas relacionadas à cirurgia, como infecção do sítio cirúrgico ou pneumonia nosocomial. Um dos mecanismos que levam à imunossupressão pós-operatória é a apoptose de células imunológicas. Durante a cirurgia, a anestesia se destina a minimizar as alterações prejudiciais e manter a homeostase perioperatória. O objetivo deste estudo foi avaliar o efeito da técnica anestésica usada para artroplastia total de joelho sobre a apoptose em linfócitos de sangue periférico no pós-operatório. MÉTODOS: Trinta e quatro pacientes submetidos à artroplastia total primária de joelho foram randomicamente designados para dois protocolos de anestesia regional: raquianestesia e bloqueio combinado raqui-peridural. Onze pacientes submetidos à artroplastia total do joelho sob anestesia geral formaram o grupo controle. Antes da cirurgia, logo após a cirurgia, durante o primeiro dia de pós-operatório e sete dias após a cirurgia, amostras de sangue venoso foram colhidas e o estado imunológico do paciente foi avaliado com o uso deflow cysts 87 m, juntamente com apoptose de linfócitos com o uso de microscopia de fluorescência. RESULTADOS: Apoptose em linfócitos de sangue periférico foi observada imediatamente no pós-operatório e acompanhada por uma redução do número de células T e B. Não houve diferença significativa no número de linfócitos apoptóticos de acordo com o protocolo anestésico. Alterações no número de células T CD3/8 e no número de linfócitos apoptóticos foram observadas no sétimo dia após a cirurgia. CONCLUSÃO: Apoptose em linfócitos de sangue periférico é um evento precoce no período pós-operatório que dura até sete dias e não é afetado pela escolha da técnica anestésica.

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.

ANTIPROLIFERATIVE EFFECTS ON BREAST CANCER CELLS AND SOME INTERACTIONS OF NEW DISTAMYCIN ANALOGUES WITH DNA, ENDONUCLEASES AND DNA TOPOISOMERASES.

The evaluation of a new group of distamycin analogues 1-6 as potential minor groove binders for the treatment of cancer were investigated. The activity of the new compounds against several restriction enzymes was examined. The studied compounds did not block GC-rich sequences regions of DNA but inhibited catalytic action of endonucleases in AA, AT, TT and AG restriction sites. Determination of association constants using calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 have confirmed that the tested compounds bind within minor groove of B-DNA. All of the compounds demonstrated activity against DNA topoisomerases II at the concentration 10 µM, but they did not inhibit activity of topoisomerase I. The studied derivatives were evaluated in human MCF-7 breast cancer cells and showed antiproliferative and cytotoxic effects in the range of 81.70 µM and 200.00 µM.

Activity of the kynurenine pathway and its interplay with immunity in patients with pulmonary arterial hypertension.

OBJECTIVE: We evaluated blood concentrations of kynurenine pathway metabolites, natural and induced regulatory T cells (nTreg, iTreg), and Th17 cells in order to examine the activity of the kynurenine pathway and its relation to immune status in patients with pulmonary arterial hypertension (PAH). METHODS: Plasma concentrations of tryptophan, kynurenine, kynurenic acid, anthranilic acid, and 3-hydroxykynurenine were quantified in 26 patients with PAH (vs 30 healthy controls) at baseline and after 6 months, and assessed them in relation to clinical parameters, frequencies of lymphocyte subsets, and outcome. RESULTS: The PAH group presented higher concentrations of tryptophan (52.9 (IQR 46.3-57.5) vs 40.3 (35.2-46.3) µmol/L, p=0.00003), kynurenine 2.8 (2.4-3.4) vs 1.9 (1.5-2.3) µmol/L, p=0.000007), kynurenine/tryptophan ratio (0.051 (0.044-0.064) vs 0.043 (0.039-0.055), p=0.03), iTreg frequencies (10.5 (8.8-13.9)% vs 6.8 (5.2-9.5)%, p=0.002) and iTreg/Th17 (1.73 (1.2-2.8) vs 0.93 (0.61-1.27), p=0.003) together with lower ratios of kynurenic acid/kynurenine, 3-hydroxykynurenine/kynurenine, and anthranilic acid/kynurenine. Kynurenine concentrations and kynurenine/tryptophan ratio correlated positively with iTreg/Th17, and inversely with Th17 subsets, whereas kynurenic acid/kynurenine and anthranilic acid/kynurenine ratios correlated positively with Th17. Adverse outcomes occurred in 9 of 26 patients and they showed higher baseline concentrations of kynurenine (3.6 (2.8-4.3) vs 2.7 (2.1-3.2) µmol/L, p=0.033). Median kynurenine values ≥3.4 µmol/L (67% sensitivity, 94% specificity) identified patients with a worse clinical course. CONCLUSIONS: PAH is characterised by upregulated tryptophan metabolism and enhanced biosynthesis of kynurenine. Elevated kynurenine concentration is associated with an adverse clinical course. Dysregulated immunity, delineated by Treg-Th17 imbalance, is directly related to diverse activation of the kynurenine pathway, indicating the potential interplay between kynurenines and the immune system in PAH.

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.