Insights into the Molecular Mechanism of Endothelial Glycocalyx Dysfunction during Heart Surgery.

The endothelial glycocalyx (EGC) is a layer of proteoglycans (associated with glycosaminoglycans) and glycoproteins, which adsorbs plasma proteins on the luminal surface of endothelial cells. Its main function is to participate in separating the circulating blood from the inner layers of the vessels and the surrounding tissues. Physiologically, the EGC stimulates mechanotransduction, the endothelial charge, thrombocyte adhesion, leukocyte tissue recruitment, and molecule extravasation. Hence, severe impairment of the EGC has been implicated in various pathological conditions, including sepsis, diabetes, chronic kidney disease, inflammatory disorders, hypernatremia, hypervolemia, atherosclerosis, and ischemia/reperfusion injury. Moreover, alterations in EGC have been associated with altered responses to therapeutic interventions in conditions such as cardiovascular diseases. Investigation into the function of the glycocalyx has expanded knowledge about vascular disorders and indicated the need to consider new approaches in the treatment of severe endothelial dysfunction. This review aims to present the current understanding of the molecular mechanisms underlying cardiovascular diseases and to elucidate the impact of heart surgery on EGC dysfunction.

Involvement of M1-Activated Macrophages and Perforin/Granulysin Expressing Lymphocytes in IgA Vasculitis Nephritis.

We investigated the polarisation of CD68+ macrophages and perforin and granulysin distributions in kidney lymphocyte subsets of children with IgA vasculitis nephritis (IgAVN). Pro-inflammatory macrophage (M)1 (CD68/iNOS) or regulatory M2 (CD68/arginase-1) polarisation; spatial arrangement of macrophages and lymphocytes; and perforin and granulysin distribution in CD3+ and CD56+ cells were visulaised using double-labelled immunofluorescence. In contrast to the tubules, iNOS+ cells were more abundant than the arginase-1+ cells in the glomeruli. CD68+ macrophage numbers fluctuated in the glomeruli and were mostly labelled with iNOS. CD68+/arginase-1+ cells are abundant in the tubules. CD56+ cells, enclosed by CD68+ cells, were more abundant in the glomeruli than in the tubuli, and co-expressed NKp44. The glomerular and interstitial/intratubular CD56+ cells express perforin and granulysin, respectively. The CD3+ cells did not express perforin, while a minority expressed granulysin. Innate immunity, represented by M1 macrophages and CD56+ cells rich in perforin and granulysin, plays a pivotal role in the acute phase of IgAVN.

Endothelial Dysfunction in Patients Undergoing Cardiac Surgery: A Narrative Review and Clinical Implications.

Cardiac surgery is one of the highest-risk procedures, usually involving cardiopulmonary bypass and commonly inducing endothelial injury that contributes to the development of perioperative and postoperative organ dysfunction. Substantial scientific efforts are being made to unravel the complex interaction of biomolecules involved in endothelial dysfunction to find new therapeutic targets and biomarkers and to develop therapeutic strategies to protect and restore the endothelium. This review highlights the current state-of-the-art knowledge on the structure and function of the endothelial glycocalyx and mechanisms of endothelial glycocalyx shedding in cardiac surgery. Particular emphasis is placed on potential strategies to protect and restore the endothelial glycocalyx in cardiac surgery. In addition, we have summarized and elaborated the latest evidence on conventional and potential biomarkers of endothelial dysfunction to provide a comprehensive synthesis of crucial mechanisms of endothelial dysfunction in patients undergoing cardiac surgery, and to highlight their clinical implications.

Human Decidual CD1a+ Dendritic Cells Undergo Functional Maturation Program Mediated by Gp96.

Heat shock proteins (hsps), in certain circumstances, could shape unique features of decidual dendritic cells (DCs) that play a key role in inducing immunity as well as maintaining tolerance. The aim of the study was to assess the binding of gp96 to Toll-like receptor (TLR) 4 and CD91 receptors on decidual CD1a+ DCs present at the maternal-fetal interface in vitro as well as the influence of CD1a+ DCs maturation status. Immunohistology and immunofluorescence of paraffin-embedded first-trimester decidua tissue sections of normal and pathological (missed abortion MA and blighted ovum BO) pregnancies were performed together with flow cytometry detection of antigens in CD1a+ DCs after gp96 stimulation of decidual mononuclear cells. Gp96 efficiently bound CD91 and TLR4 receptors on decidual CD1a+ DCs in a dose-dependent manner and increased the expression of CD83 and HLA-DR. The highest concentration of gp96 (1000 ng/mL) increased the percentage of Interferon-γ (INF-γ) and IL-15 expressing gp96+ cells. Gp96 binds CD91 and TLR4 on decidual CD1a+ DCs, which causes their maturation and significantly increases INF-γ and IL-15 in the context of Th1 cytokine/chemokine domination, which could support immune response harmful for ongoing pregnancy.

Predictive Value of Monocyte Chemoattractant Protein-1 in the Development of Diastolic Dysfunction in Patients with Psoriatic Arthritis.

We aimed to evaluate the diagnostic accuracy of the proinflammatory monocyte chemotactic protein-1 (MCP-1) in the diagnosis of asymptomatic diastolic dysfunction (DD) in patients with psoriatic arthritis (PsA). The disease activity in psoriatic arthritis (DAPSA) was determined using clinical and laboratory parameters, and echocardiography was performed to estimate DD. Serum MCP-1 concentrations were elevated in PsA patients with DD diagnosed with ultrasound (median (25th percentile, 75th percentile): 366.6 pg/mL (283, 407.1 pg/mL) vs. 277.5 pg/mL (223.5, 319.1 pg/mL) in controls; P < 0.0017). PsA patients with serum MCP-1 concentration higher than the cut-off value of 347.6 pg/mL had a 7.74-fold higher chance of developing DD than PsA patients with lower serum MCP-1 concentrations (controls), with a specificity of 86.36% and sensitivity of 55%, as verified using ultrasound. The group with MCP-1 concentrations above the cut-off value also showed a higher late peak diastolic mitral inflow velocity, A-wave value (P = 0.000005), E/E' ratio (P = 0.00005), and a lower E/A ratio (P = 0.000002), peak systolic left atrial reservoir strain, SA value (P = 0.0066), early peak diastolic displacement of the mitral septal annulus, E' wave value (P = 0.003), than controls. Systolic blood pressure (P = 0.01), LDL cholesterol concentration (P = 0.012), glucose concentration (P = 0.011), and DAPSA (P = 0.0000) increased in the PsA group with higher MCP-1 concentrations, although there were no differences in comorbidities and therapy between the groups compared. Thus, the serum MCP-1 concentration was a significant and independent prognostic indicator for asymptomatic DD in PsA patients (area under the curve = 0.730, P = 0.001). The DAPSA score in PsA patients might indicate the need for echocardiography and adjustment of anti-inflammatory treatment in terms of DD prevention.

Granulysin expression and granulysin-mediated apoptosis in the peripheral blood of osteoarthritis patients.

Osteoarthritis (OA) is a chronic joint disease caused by mechanical damage and metabolic factors that support the development of low-grade inflammation. Increased levels of T helper 1 pro-inflammatory cytokines in the serum of OA patients may support granulysin (GNLY) mediated cytotoxicity, which in-turn may contribute to the pathogenesis of OA. In the present study, GNLY expression and cytotoxic/apoptotic mechanisms mediated by GNLY in the peripheral blood of OA patients were assessed. A total of 40 non-obese women (median age of 64 years old) with knee OA, and 40 controls (median age 62 years old) were enrolled in the study. GNLY, IFN-γ and IL-4 expression levels were investigated in peripheral blood lymphocytes (PBLs) using flow cytometry, immunocytochemistry and/or confocal microscopy. Natural killer (NK) GNLY-mediated apoptosis through NK effectors against K-562 targets was analyzed using the PKH-26 18-h cytotoxicity assay. Serum GNLY levels were assessed using ELISA. The percentage of GNLY+PBLs was higher in the OA patients than that in the controls due to the increase in the proportions of GNLY+ cells in the natural killer (NK), T and natural killer T (NKT) subsets. GNLY localization inside exocytotic lysosomal-associated membrane protein-1+ granules was ~40% in both groups. However, the intensity of GNLY labeling in PBLs was higher in OA patients than in the controls, and it was supported by the increased expression of IFN-γ relative to IL-4 in NK and T cells from OA patients. The serum GNLY concentration was <0.3 ng/ml in both groups. RC8 anti-GNLY mAb by itself was unable to significantly alter early apoptosis, whereas RC8 anti-GNLY mAb combined with anti-perforin mAb significantly reduced NK-mediated early apoptosis of K-562 targets in the OA patients, whilst not exerting a notable effect in the controls. Anti-perforin mAb by itself did not affect apoptosis significantly. These results suggest that in women with knee OA, GNLY expression in the PBL subsets and GNLY-mediated early apoptosis of K-562 targets are increased compared with the controls and accompanied by intracellular dominance of IFN-γ over IL-4 in NK cells.

Persistence and Severity of Cutaneous Manifestations in IgA Vasculitis Is Associated with Development of IgA Vasculitis Nephritis in Children.

BACKGROUND/OBJECTIVE: The purpose of this study was to evaluate the prevalence and severity of skin involvement in children with IgA vasculitis (IgAV) and its relationship with clinical and biochemical parameters and the risk of developing IgA vasculitis nephritis (IgAVN), the only cause of long-term morbidity and the main prognostic factor in IgAV patients. METHODS: This national multicenter retrospective study included 611 patients under the age of 18 years with IgAV referred to five Croatian tertiary hospitals between 2009 and 2019. Patient data were collected from a database with systematic analysis of IgAV patients in the Croatian population. RESULTS: Among the 611 children, 205 (33.55%) had purpura on the lower extremities, in 207 (33.88%) the rash extended on the trunk, in 149 (24.39%) it extended to the upper extremities, in 32 (5.24%) the rash was generalized, while 15 (2.47%) had the most severe skin symptoms: bullae, ulcerations, and necroses. IgAVN developed in 130 (21.28%) and persistent IgAVN (present for >3 months) in 48 (7.86%) children. Multivariate logistic regression found that presence of ulcerations and necroses (OR 3.20 [95% CI 1.03-9.91]), persistent purpura (OR 2.89 [95% CI 1.71-4.88]), and higher age (OR 1.16 [95% CI 1.09-1.23]) were significant predictors of IgAVN, whereas persistent purpura (OR 20.11 [95% CI 1.09-372.52]), male sex (OR 3.32 [95% CI 1.13-9.80]), and higher age (OR 1.15 [95% CI 1.00-1.30]) were predictors of persistent IgAVN. Among the laboratory parameters, higher serum urea (OR 1.43 [95% CI 1.03-2.00]) and reduction in activated partial thromboplastin time (OR 0.83 [95% CI 0.74-0.93]) were shown to have a significant impact on increasing the risk of persistent IgAVN. CONCLUSION: With increasing severity and duration of cutaneous manifestations in IgAV, the risk of developing IgAVN increases, making the prognosis worse, with a greater likelihood to need more aggressive treatment.

Gastrointestinal involvement and its association with the risk for nephritis in IgA vasculitis.

BACKGROUND: We analysed clinical and biochemical parameters in predicting severe gastrointestinal (GI) manifestations in childhood IgA vasculitis (IgAV) and the risk of developing renal complications. METHODS: A national multicentric retrospective study included children with IgAV reviewed in five Croatian University Centres for paediatric rheumatology in the period 2009-2019. RESULTS: Out of 611 children, 281 (45.99%) had at least one GI manifestation, while 42 of 281 (14.95%) had the most severe GI manifestations. Using logistic regression several clinical risk factors for the severe GI manifestations were identified: generalized rash [odds ratio (OR) 2.09 (95% confidence interval (CI) 1.09-4.01)], rash extended on upper extremities (OR 2.77 (95% CI 1.43-5.34)] or face [OR 3.69 (95% CI 1.42-9.43)] and nephritis (IgAVN) [OR 4.35 (95% CI 2.23-8.50)], as well as lower values of prothrombin time (OR 0.05 (95% CI 0.01-0.62)], fibrinogen [OR 0.45 (95% CI 0.29-0.70)] and IgM [OR 0.10 (95% I 0.03-0.35)]] among the laboratory parameters. Patients with severe GI involvement more frequently had relapse of the disease [OR 2.14 (CI 1.04-4.39)] and recurrent rash [OR 2.61 (CI 1.27-5.38)]. Multivariate logistic regression found that the combination of age, GI symptoms at the beginning of IgAV and severity of GI symptoms were statistically significant predictors of IgAVN. Patients in whom IgAV has started with GI symptoms [OR 6.60 (95% CI 1.67-26.06)], older children [OR 1.22 (95% CI 1.02-1.46)] with severe GI form of IgAV (OR 5.90 (95% CI 1.12-31.15)] were particularly high-risk for developing IgAVN. CONCLUSION: We detected a group of older children with the onset of GI symptoms before other IgAV symptoms and severe GI form of the IgAV, with significantly higher risk for acute and chronic complications of IgAV.

Decrease of Perforin Expressing Lymphocytes after On-Pump Coronary Artery Bypass Grafting Surgery Irrespective of Carbohydrate Preoperative Oral Feeding.

BACKGROUND: Coronary artery bypass grafting (CABG) surgery continues to be the gold standard for treating the patients with coronary artery disease. CABG surgery can be performed on or off cardiopulmonary bypass, termed as on-pump or off-pump CABG, respectively. It has been shown that CABG surgery, preferably on-pump CABG surgery, leads to the changes of cell immunity during perioperative and early postoperative period. The mechanisms of regulation of the immune response in patients during and early after surgical revascularization are not fully understood. The aim of this study was to investigate the influence of carbohydrate preoperative oral feeding on frequency and perforin expression in peripheral blood lymphocytes in patients after on- or off-pump CABG surgery in early postoperative period. PATIENTS AND METHODS: In this prospective clinical study, 80 patients scheduled for CABG surgery were included in the study. The patients were randomly allocated into four groups (20 in each group): patients in Group 1 underwent on-pump CABG and did not receive carbohydrate preoperative oral feeding; patients in Group 2 underwent on-pump CABG and were preoperatively fed; patients in Group 3 underwent off-pump CABG and did not receive carbohydrate preoperative oral feeding; while patients in Group 4 underwent off-pump CABG and received carbohydrate preoperative oral feeding. Blood samples were collected immediately before (T1), 24 (T2) and 72 (T3) hours after the surgery. Peripheral blood mononuclear cells were isolated by gradient centrifugation and simultaneously labelled by antigens using fluorochrome-conjugated monoclonal antibodies. Frequency of T lymphocytes, NK and NKT cells, their subsets as well as their perforin expression were detected, and analyzed by flow cytometry. RESULTS: There was significant decrease in frequency of CD3+ and CD3+CD4+ cells, as well as perforin expressing CD3+CD8+ cells in patients who underwent on-pump CABG in comparison to patients who underwent off-pump CABG 24 hours after the surgery. Carbohydrate preoperative oral feeding did not effect changes in lymphocytes subpopulations and perforin expression at any time point. CONCLUSION: Decreases of CD3+ cells on account of CD3+CD4+ subsets, and perforin expressing cells on account of CD3+CD8+ perforin+ cells were found in patients who had undergone on-pump CABG, but not in patients who had undergone off-pump CABG surgery, irrespectively of carbohydrate preoperative oral feeding.

Possible role of circulating endothelial cells in patients after acute myocardial infarction.

Acute myocardial infarction (AMI) occurs as a result of insufficient myocardial perfusion leading to cell necrosis. This is most commonly due to the obstruction of the coronary artery by ruptured atherosclerotic plaque and thrombosis. Damaged ischemic and necrotic myocardial cells release pro-inflammatory substances in tissue and plasma, leading to a systemic inflammatory response. Profound systemic inflammatory response during ischemia/reperfusion injury causes disruption of endothelial glycocalyx and detachment of endothelial cells that express von Willebrant factor (vWF). We hypothesize that circulating vWF+ endothelial cells could act as antigen presenting cells which interact with T and NK cells directly, by cell to cell contact and indirectly by cytokine and chemokine secretion, leading to the immune response towards inflammation. Analyzing the frequency, phenotype and pro-inflammatory substances produced in circulating vWF positive (+) cells in patients with AMI could be beneficial to determine the severity of the pro-inflammatory response, according to the level of endothelial dysfunction in the early period of AMI. To evaluate these hypotheses, we suggest to determine frequency, phenotype, and ability of cytokine/chemokine production in circulating vWF+ endothelial cells by simultaneous surface and intracellular cell staining, and flow cytometry analysis. Secretion of pro-inflammatory cytokines and chemokines, pro-atherogenic substances and the components of glycocalyx might be measured in supernatants of magnetically separated or sorted vWF+ endothelial cells, as well as in the serum of a patient with acute AMI by enzyme linked-immunoassay tests. The interaction of increasing concentrations of isolated circulating vWF+ endothelial cells and cognate T and NK cells might be investigated by lymphocyte proliferation rate, cytotoxic mediators' expression, and cytokine production. If our hypothesis is correct, characterization of circulating vWF+ endothelial cells could grant us greater insight into their role in pathophysiology of AMI and the degree of myocardial damage.

Assessing whether progesterone-matured dendritic cells are responsible for retention of fertilization products in missed abortion.

We hypothesize that progesterone causes tolerogenic maturation of myeloid dendritic cells (DCs) in human decidua of threatened miscarriage or missed abortion characterized by a distinct phenotype and cytokine production, including reduction of the main NK cell proliferation and cytotoxic factor interleukin (IL)-15. During DC/NK cell interaction, progesterone-shaped DCs cannot efficiently multiply or equip NK cells with the cytotoxic mediators peforin and granulysin, which might harm trophoblasts and induce abortion. We propose that the presence, and maturation stage of decidual myeloid DCs be investigated using semi-quantitative immunohistological analyses and/or double-color immuno-fluorescent labeling of DC lineage and activation markers. The spatial arrangement of granulysin+ cells, NKp46+ NK cells, DCs, and trophoblasts might provide information about their mutual interactions in vivo. Multiple flow cytometry analyses of NK-receptors would provide insight into NK cell activation status. NK cell activation status could be also assessed by cytotoxicity assays against trophoblast cell lines, or isolated cognate extra-villous trophoblast cells. A correlation between decidual progesterone concentration or IL-15 expression, and the degree of DC maturation or the frequency of granulysin+ cells, might help to elucidate the mechanism of abortion retention in utero.

Granulysin-mediated apoptosis of trophoblasts in blighted ovum and missed abortion.

PROBLEM: Granulysin (GNLY) is a cytotoxic molecule mostly present in decidual natural killer (NK) cells. Blighted ovum (BO) and missed abortion (MA) represent the early pathological pregnancies with hindered development of the embryoblast or a dead embryo. We investigated the GNLY-mediated apoptotic mechanism potentially responsible for delayed termination of pregnancy. METHOD OF STUDY: We performed immunohistological and immunofluorescence labeling of decidual tissues (GNLY, Apaf-1, NF-κB). NKG2A expression was analyzed by flow cytometry and GNLY mRNA by RT-qPCR. RESULTS: The GNLY labeling intensity (H score) was lower in the nuclei of trophoblast cells in BO and MA. GNLY gene levels were inversely detected in BO and MA. A decreased decidual NK cell percentage was found in MA. NK cells from pathological pregnancies expressed lower NKG2A levels. The highest frequency of Apaf-1 was found in trophoblast cells of MA. NF-kB was highly expressed in decidual cells of BO. CONCLUSION: The reduced activation of GNLY-mediated killing might be implicated in the slower rejection of trophoblast cells in BO and MA. A decreased authentic decidual NK cell number could be responsible for low cytotoxicity against trophoblast cells in MA. In BO, trophoblast cells have a higher survival potential due to increased NF-kB expression.

Correlation between immunological-inflammatory markers and endothelial disfunction in the early stage of coronary heart disease.

Classical risk factors for endothelial dysfunction (ED), such as age, gender, total cholesterol, high-density lipoprotein cholesterol, systolic blood pressure, and smoking history are utilised for the Framingham score and Systemic Coronary Risk Estimation (SCORE) for evaluation of the 10-year cardiovascular risk in routine practice. Nonetheless, pro-inflammatory mediators are deeply involved in the initiation and the progression of ED and coronary artery disease (CAD), and act additionally or independently of metabolic factors before clinical manifestations of the disease appear. C-reactive protein, a marker of intimal thickening of the myeloid-related protein 8/14 heterodimer, monocyte chemotactic protein 1, interleukin-15, the cytotoxic mediator, granulysin, and the matrix metalloproteinase 9 could be valuable, single, fast, and non-invasive laboratory tools for ED deterioration degree assessment. We propose to investigate the impact of pro-inflammatory biomarkers on ED, measured by previously established clinical methods in patients with yet undiagnosed CAD and at medium risk for an acute coronary event. It could be useful to measure and correlate the concentration of particular inflammatory markers in peripheral blood samples and the results of the Framingham and SCORE charts, multi-slice computed tomography coronary angiography, echocardiography, brachial artery flow-mediated dilatation, carotid-femoral pulse wave velocity, ankle-brachial index, carotid wall thickening, myocardial perfusion scintigraphy, and particularly, cardiac magnetic resonance imaging. The goal would be that the degree of correlation between particular inflammatory markers and the results of some methods for the assessment of ED or cardiac ischaemic imaging could be emphasised and pro-inflammatory markers positioned in the pathogenetic algorithm of CAD.

Endothelial dysfunction mediated by interleukin-18 in patients with ischemic heart disease undergoing coronary artery bypass grafting surgery.

When medication management or percutaneous coronary intervention is not successful in patients with advanced ischemic heart disease, surgical revascularisation-predominantly coronary artery bypass grafting (CABG)-is considered the gold standard. However, CABG surgery can lead to ischemia/reperfusion injury, which is characterized by a strong inflammatory response. Interleukin (IL)-18, is a strong inflammatory mediator, that is released from cardiomyocytes and can be found in the systemic circulation of patients during and immediately after CABG surgery. The existing damage of endothelial glycocalyx in patients with ischemic heart disease is further impaired concurrently during the surgery due to the anaesthesia-surgical technique used and intravascular fluid loading. This results in the increased incidence of adverse events, including myocardial infarction. IL-18 leads to the activation of lymphocyte cytotoxicity via cytotoxic mediators (Fas ligand, Tumour necrosis factor (TNF)-related apoptosis-inducing ligand, perforin, and granulysin). We hypothesize that IL-18 is released locally in the heart and the systemic circulation in patients undergoing CABG surgery and may be correlated with the level of activity of circulating lymphocytes. In turn, this may lead to lymphocyte-mediated cytotoxicity directed toward damaged and activated endothelial cells. Shear stress glycocalyx, as well as damaged and activated endothelial cells then become the main the source of pro-inflammatory cytokines, chemokines, and adhesion molecules. These attract activated lymphocytes to adhere to the endothelium or enter the subintimal layer, increasing existing or initiating the formation of new plaques, which leads to the development of myocardial infarction during or shortly after surgery. To evaluate our hypothesis, we will measure the local concentration of IL-18 in the sinus coronarius and systemic circulation. These values will then be correlated with immunological and biochemical parameters, predominantly with the concentration of degradation products of glycocalyx and cytotoxic mediators in activated lymphocytes. If our hypothesis is correct, measuring the IL-18 concentration that is responsible for glycocalyx deterioration, may become a useful tool for predicting myocardial infarction occurrence in patients undergoing CABG surgery.

Can pain intensity in osteoarthritis joint be indicator of the impairment of endothelial function?

We propose that pathological remodeling in joint tissues of osteoarthritis (OA) patients persistently stimulates local secretion of pro-inflammatory mediators, which overflow into the blood, activating leukocytes that impair endothelial function and accelerate the atherosclerotic process. During periods of pain, endothelial dysfunction progresses more aggressively due to elevated secretion of these pro-inflammatory mediators, which are involved in both atherosclerosis and the sensation of pain. Concentrations of pro-inflammatory cytokines and their antagonists, activating and decoy receptors of the broad interleukin (IL)-1 and IL-17 families, IL-15, and monocyte chemotactic protein-1 should be measured in peripheral blood samples of OA patients and compared with (I) OA clinical severity; (II) subclinical parameters of atherosclerosis; (III) ischemic heart disease risk factors; (IV) soluble factors indicating endothelial dysfunction; (V) degree of bone destruction; and (VI) results of a six-minute walk test. Arthroscopy and joint replacement surgery provide an opportunity to estimate mRNA and protein expression of inflammatory mediators in specimens of synovial fluid, synovial membrane, cartilage, and/or subarticular bone. A range of methods, including questionnaires, X-ray, computed tomography, ultrasound, enzyme-linked immunosorbent assay, immunohistology, immunofluorescence, and reverse transcription and in situ polymerase chain reaction are available. Understanding the inflammatory and immune mechanisms underlying OA may allow the early identification of patients at high risk of cardiovascular disease, independently of classical coronary risk factors. Pain may constitute an extrinsic indicator of currently worsening endothelial function.

Potential role of heat-shock protein 70 and interleukin-15 in the pathogenesis of threatened spontaneous abortions.

PROBLEM: The role of HSP70 and both its constitutive (Hsc) and inducible (Hsp) forms in the pathogenesis of threatened spontaneous abortions was investigated. METHOD OF STUDY: Immunohistology and/or immunofluorescence was used to analyze paraffin-embedded tissue sections, and reverse transcriptase-quantitative polymerase chain reaction and flow cytometry were used for analyses of decidual mononuclear cells (DMCs) and confocal microscopy for the detection of perforin, granulysin, and lysosome-associated membrane protein-1 (LAMP-1) in decidual lymphocytes (DLs). RESULTS: The percentage of single Hsp70(+) , Hsc70(+) , and IL-15(+) cells and mRNA levels of HSP70, CD91, and TLR4 were lower in the decidua basalis in cases of threatened miscarriages compared to that in cases of normal pregnancy. In a suspension of normal DMCs, IL-15 significantly decreased the HSP70 members and TLR4 in dendritic cells, T cells, and NK cells while increasing CD91 in NK cells alone. CONCLUSION: Downregulation of Hsc70, Hsp70, and IL-15 expression at gene and/or protein levels might support the retention of fertilization products in cases of missed abortion and blighted ovum.

Colocalization of Granulysin Protein Forms with Perforin and LAMP-1 in Decidual Lymphocytes During Early Pregnancy.

PROBLEM: Granulysin (GNLY) occurs in two forms, which have molecular weights of 9 and 15 kDa. We analyzed the cytotoxic potential of decidual lymphocytes (DLs) and peripheral blood lymphocytes (PBLs) based on the forms of GNLY that colocalizes with perforin (PER) and LAMP-1 following activation. METHODS: The forms of GNLY were detected by using confocal microscopy. We investigated the colocalization with PER and LAMP-1 in freshly isolated and activated DLs and PBLs. RESULTS: Activation of DLs and PBLs by K-562 cells increased the colocalization of 9 kDa GNLY with PER and LAMP-1. K-562 cells transfected with HLA-C decreased 9 kDa GNLY colocalization with PER in DLs only. IL-15 in DLs decreased 9 kDa GNLY and LAMP-1 colocalization, but increased both 15 kDa GNLY and LAMP-1, and PER and LAMP-1 colocalization. CONCLUSION: Activated DLs and PBLs show greater cytotoxic potential based on increased colocalization of 9 kDa GNLY and PER. HLA-C and IL-15 affect DLs, indicating their role in maintaining the pregnancy tolerance.

Possible role of granulysin in pathogenesis of osteoarthritis.

Increased presence of immune mediator and cytotoxic/apoptotic molecule granulysin was noticed in different tissues during pathological processes with the domination of Th1 over Th2 mediated immunity. Beside granulysin expression in T and NKT cells, activated NK cells are thought to be the major source of chemotactic 15 kDa and cytotoxic 9 kDa granulysin in vivo. As NK cells are the principal joint's tissue-infiltrating lymphocyte subset, we hypothesized that granulysin mediated human cell death (apoptosis) could be responsible for the relatively silent damage of the joint's tissue without clinically notable signs of systemic inflammation in the patients with osteoarthritis (OA). The analyzes of the presence and frequency of granulysin expressing lymphocytes at protein and gene levels in peripheral blood and synovial samples and/or the samples of joint's tissue after the joint replacement therapy in patients with OA could give the initial insight to evaluate our hypothesis. It would be of the particular interest to differentiate the expression of 9 kDa and 15 kDa granulysin forms in the effector cells, since only the shorter form exhibits cytotoxic properties. The measurement of granulysin mediated early apoptosis in human NK sensitive K562 cells could be suitable in vitro model for evaluating granulysin activity. Furthermore, disturbed balance of pro-inflammatory and anti-inflammatory cytokines in OA patients, could influence the level of the granulysin expression. Having in mind that the granulysin and its regulation is still unknown in the pathogenesis of OA, it could be worth to explore this important pro-inflammatory, cytotoxic/apoptotic mediator.

Role of tumor-associated glycoprotein-72 in the progression of endometrial adenocarcinoma: a proposed study.

Endometrial adenocarcinoma is on the basis of the molecular, immunohistological and clinicopathologic features broadly divided into two groups, referred as type I and type II. Type I appears more frequently and in principle patients have a good prognosis; however a significant number of patients develop local recurrences. We hypothesize that TAG-72, expressed on endometrial carcinoma binds and internalizes endocytic pattern recognition receptors on surrounding tissue antigen presenting cells (dendritic cells and macrophages), powers their anti-inflammatory maturation program and make them capable to elicit or modulated tolerogenic immune response mediated by local T and NK effectors. This could support uncontrolled local tumor growth, deeper tumor invasion into surrounding tissues, frequent local recurrences and/or lymph node metastasis. To test this hypothesis, we propose a semi-quantitative immunohistochemical analysis of TAG-72 expression in endometrial adenocarcinoma samples and to correlate the results with clinical and pathological parameters (age, type and histological grade of the tumor, estrogen and progesterone receptor expression, invasion into the myometrium and capillaries, presence of lymph node metastases, FIGO stage, and TNM classification). It would be worthwhile to investigate the local tissue immune response in the tumor environment using tissue samples removed during surgery. These studies could elucidate the underlying immunopathological mechanisms that govern the early recurrence and possibly distant metastases of TAG-72-expressing adenocarcinomas and might help in deciding the type of treatment to be applied in a selected group of cancer patients including application of biological therapy with anti-TAG-72 antibodies, according the principle of personalized oncology treatments.

Heat-Shock Proteins 70 Induce Pro-Inflammatory Maturation Program in Decidual CD1a(+) Dendritic Cells.

PROBLEM: The aim of the study was to assess possible binding of a mixture of constitutive Hsc70 and inducible Hsp70 forms (HSP70) to Toll-like receptor (TLR) 4 and CD91 receptors on decidual CD1a(+) dendritic cells (DCs) and their influence on DCs maturation status. METHOD OF STUDY: Immunohistology and immunofluorescence of paraffin-embedded first trimetester and term pregnancy decidua were performed together with flow cytometry detection of antigens in DCs after stimulation of decidual mononuclear cells with HSP70. RESULTS: Hsc70 and Hsp70 labeling revealed intracellular and nuclear staining in trophoblast cells. The numbers of Hsc70(+) and Hsp70(+) cells of decidual tissue were higher in early pregnancy decidua than in decidua at term. HSP70 binds CD91 and TLR4 receptors on CD1a(+) DCs and increased the expression of CD83, HLA-DR, CD80, and CD86, but decreased CC receptor (CCR) 5. HSP70 increased CC ligand (CCL) 3 and CCL22. HSP70 in the concentration of 1 µg/mL increased the percentage of interferon-γ and interleukin (IL)-15-expressing cells over the cells expressing IL-4. CONCLUSION: HSP70 binds CD91 and TLR4 on decidual CD1a(+) DCs, causes their maturation, and increases IL-15 in the context of Th1 cytokine/chemokine domination, which could support immune response harmful for ongoing pregnancy.