The Biological Role of Pleural Fluid PAI-1 and Sonographic Septations in Pleural Infection: Analysis of a Prospectively Collected Clinical Outcome Study.

Rationale: Sonographic septations are assumed to be important clinical predictors of outcome in pleural infection, but the evidence for this is sparse. The inflammatory and fibrinolysis-associated intrapleural pathway(s) leading to septation formation have not been studied in a large cohort of pleural fluid (PF) samples with confirmed pleural infection matched with ultrasound and clinical outcome data. Objectives: To assess the presence and severity of septations against baseline PF PAI-1 (Plasminogen-Activator Inhibitor-1) and other inflammatory and fibrinolysis-associated proteins as well as to correlate these with clinically important outcomes. Methods: We analyzed 214 pleural fluid samples from PILOT (Pleural Infection Longitudinal Outcome Study), a prospective observational pleural infection study, for inflammatory and fibrinolysis-associated proteins using the Luminex platform. Multivariate regression analyses were used to assess the association of pleural biological markers with septation presence and severity (on ultrasound) and clinical outcomes. Measurements and Main Results: PF PAI-1 was the only protein independently associated with septation presence (P < 0.001) and septation severity (P = 0.003). PF PAI-1 concentrations were associated with increased length of stay (P = 0.048) and increased 12-month mortality (P = 0.003). Sonographic septations alone had no relation to clinical outcomes. Conclusions: In a large and well-characterized cohort, this is the first study to associate pleural biological parameters with a validated sonographic septation outcome in pleural infection. PF PAI-1 is the first biomarker to demonstrate an independent association with mortality. Although PF PAI-1 plays an integral role in driving septation formation, septations themselves are not associated with clinically important outcomes. These novel findings now require prospective validation.

Impact of low-level laser therapy as an adjunct to non-surgical periodontal treatment on the levels of tissue plasminogen activator and plasminogen activator inhibitor 1 in Stage 3-4, Grade C periodontitis patients: a split-mouth, randomized control study.

AIM: To investigate the effects of low-level laser therapy (LLLT) as an adjunct to non-surgical periodontal treatment (NSPT) on the plasminogen-activating system. MATERIALS AND METHODS: Stage 3-4 Grade C periodontitis and age-gender-matched healthy individuals participated in the split-mouth study (ClinicalTrials.gov identifier, NCT05233501). The study groups were Periodontitis/NSPT (Sham); Periodontitis/NSPT + LLLT (LLLT); Healthy (Control). Following NSPT, LLLT was applied on Days 0, 2 and 7. Clinical parameters were recorded at baseline and on Day 30. Gingival crevicular fluid (GCF) was collected at baseline, on days 7, 14, and 30; tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) levels were measured with ELISA. RESULTS: Clinical parameters, total GCF tPA (tPAt) and PAI-1 (PAI-1t) levels significantly reduced in LLLT and Sham groups (< 0.001). GCF tPAt levels in LLLT were significantly lower (< 0.05) than Sham on Day 7. GCF tPAt levels in periodontitis groups were significantly higher than the Control at baseline, on Days 7 and 14 (< 0.01). By Day 30, both groups decreased to control levels (> 0.05). GCF PAI-1t levels were significantly lower in LLLT than the Sham on day 30 (< 0.01), comparable to healthy controls (> 0.05). CONCLUSION: Adjunctive LLLT modulates the plasminogen activating system in severe periodontitis by altering GCF tPA and PAI-1 levels. CLINICAL RELEVANCE: LLLT as an adjunct to non-surgical periodontal treatment in patients with Stage 3-4 Grade C leads to reduced plasminogen activation.

[Biochemical markers of thrombotic complications in the acute period of ischemic stroke].

AIM: To study the profile of biochemical markers of the hemostasis system, to clarify their role and relationships in the pathogenesis of the development of thrombotic complications (TC) of ischemic stroke (IS) and the associated assessment of the possibilities of their diagnostic application. MATERIALS AND METHODS: The study group included 302 patients (164 men, 138 women) who were admitted to the hospital with a diagnosis of IS within 24 hours of the onset of the disease. The diagnosis was confirmed by computed tomography. The average age of patients was 69 (5088) years. Blood was taken from all patients on the 1st day of the disease to determine the profile of analytes presumably associated with the pathogenesis of TC. Levels of homocysteine, protein C inhibitor, thrombomodulin, plasminogen, tissue plasminogen activator, urokinase, plasminogen activator type 1 inhibitor, t-PA/PAI-1 complex, vitronectin, plasmin-2-antiplasmin complex, D-dimer, fibronectin were determined in blood serum by ELISA. RESULTS: TC in the acute period of IS (up to 21 days) were recorded in 32 (10.6%, 95% CI 7.3714.3) patients, of which pulmonary embolism was observed in 27 (8.94%, 95% CI 5.9812.4) patients, deep vein thrombosis in 5 (1.66%, 95% CI 0.473.47) patients. The results of the study of a panel of specific proteins involved in pathogenetic processes accompanying necrosis of brain tissue in IS demonstrated that of the entire list of markers of the hemostasis system activation selected for the study, the most significant are: the concentration of fibronectin in the prognosis of the absence of TC with a threshold value of more than 61 mkg/ml and OR 4.4 (95% CI 1.512.9, p=0.011), and the concentration of the t-PA/PAI-1 complex in the prognosis of the development of TC with a threshold value of more than 14 ng/ml and OR 11.3 (95% CI 1.18109.3, p=0.03). CONCLUSION: The significance of the t-PA/PAI-1 complex and fibronectin as markers of TC in IS may be due to a violation of the activation processes of the fibrinolytic link of hemostasis and the accumulation of non-deposited compounds that damage the vascular wall.

Biological mechanisms and individual variation in fibrinolysis after major trauma.

OBJECTIVE: To understand more about the individual variation in the time course of fibrinolysis following major injury and to assess the potential for stratification of trauma patients for tranexamic acid (TXA) therapy. METHODS: A historical dataset (from 2004) was used, consisting of samples from 52 injured patients attended by a medical prehospital system. Blood samples were taken at the incident scene, on arrival in the emergency department, 2.5 hours after hospital arrival and 5 hours after hospital arrival. From the study database, we extracted values for tissue-type plasminogen activator (tPA; an activator of fibrinolysis), one of the plasminogen activator inhibitors (PAI-1; as a natural inhibitor of fibrinolysis) and D-dimer (as a marker of the extent of fibrinolysis). RESULTS: The changes over time in median tPA and PAI-1 were mirror images, with initial high tPA levels which then rapidly decreased and low initial PAI-1 levels which slowly increased. There were high levels of fibrinolytic activity (D-dimer) throughout. This pattern was present in patients across a broad range of injury severities. CONCLUSIONS: After major trauma, there seems to be an early 'antifibrinolytic gap' with the natural antifibrinolytic system lagging several hours behind the natural profibrinolytics. An early dose of exogenous antifibrinolytic (TXA) might have its effect by filling this gap. The finding that tPA and subsequent clot breakdown (illustrated by D-dimer formation) are raised in a broad range of patients, with little correlation between the initial fibrinolytic response and markers of injury severity, may be the reason that TXA is effective across a broad range of injured patients.

Low concentrations of cadmium chloride promotes protein translation and improve cell line productivity.

Protein translation has emerged as a critical bottleneck for overall productivity of biological molecules. An augmentation of protein translation can be achieved by cell line engineering or by sophisticated vector design. However, for industrial process development purposes, identification of media additives that promote translation will be of great value, obviating the generation of new host platforms. Here, we examined the effect of low cadmium chloride concentrations on protein synthesis and cell line productivity. At low micromolar concentrations, cadmium chloride induced the mTOR pathway and promoted total protein synthesis in HEK 293T and CHO-K1 cells with minimal toxicity. In a parallel screening of kinase inhibitors for promoting protein expression, we identified the RSK1 inhibitor, BI-D1870, as having a transcription promoting activity on cytomegalovirus promoter-driven transgenes. Fed-batch analyses of CHO-K1 cells producing the anticoagulant factor tissue plasminogen activator (tPA) demonstrated that inclusion of cadmium chloride alone and particularly in combination with BI-D1870 improved overall yields of tPA by more than two-fold with minimal effect on cell growth. We, therefore, underscore the use of cadmium alone and in combination with BI-D1870 for improving bioproduction yields.

Intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation.

BACKGROUND: Intravenous fluids, an essential component of sepsis resuscitation, may paradoxically worsen outcomes by exacerbating endothelial injury. Preclinical models suggest that fluid resuscitation degrades the endothelial glycocalyx, a heparan sulfate-enriched structure necessary for vascular homeostasis. We hypothesized that endothelial glycocalyx degradation is associated with the volume of intravenous fluids administered during early sepsis resuscitation. METHODS: We used mass spectrometry to measure plasma heparan sulfate (a highly sensitive and specific index of systemic endothelial glycocalyx degradation) after 6 h of intravenous fluids in 56 septic shock patients, at presentation and after 24 h of intravenous fluids in 100 sepsis patients, and in two groups of non-infected patients. We compared plasma heparan sulfate concentrations between sepsis and non-sepsis patients, as well as between sepsis survivors and sepsis non-survivors. We used multivariable linear regression to model the association between volume of intravenous fluids and changes in plasma heparan sulfate. RESULTS: Consistent with previous studies, median plasma heparan sulfate was elevated in septic shock patients (118 [IQR, 113-341] ng/ml 6 h after presentation) compared to non-infected controls (61 [45-79] ng/ml), as well as in a second cohort of sepsis patients (283 [155-584] ng/ml) at emergency department presentation) compared to controls (177 [144-262] ng/ml). In the larger sepsis cohort, heparan sulfate predicted in-hospital mortality. In both cohorts, multivariable linear regression adjusting for age and severity of illness demonstrated a significant association between volume of intravenous fluids administered during resuscitation and plasma heparan sulfate. In the second cohort, independent of disease severity and age, each 1 l of intravenous fluids administered was associated with a 200 ng/ml increase in circulating heparan sulfate (p = 0.006) at 24 h after enrollment. CONCLUSIONS: Glycocalyx degradation occurs in sepsis and septic shock and is associated with in-hospital mortality. The volume of intravenous fluids administered during sepsis resuscitation is independently associated with the degree of glycocalyx degradation. These findings suggest a potential mechanism by which intravenous fluid resuscitation strategies may induce iatrogenic endothelial injury.

Type 2 Diabetes and Cardiovascular Factors Contrasted with Fibrinolysis Disorders in the Blood of Patients with Peripheral Arterial Disease.

Background and objectives: Both in the pathogenesis of type 2 diabetes (DM 2) and Peripheral Arterial Disease (PAD), a vital role is played by endothelial dysfunction. Metabolic disorders found in DM 2 (hyperglycemia, insulin resistance), endothelial dysfunction, and increased inflammation lead to intensified atherothrombosis. The fibrinolysis system comprises a natural compensatory mechanism in case of hypercoagulability. The aim of this study was to assess concentrations of selected fibrinolysis parameters in the blood of patients with symptomatic PAD, including in particular concurrent DM 2 and other cardiovascular factors. Materials and Methods: In the group of 80 patients with PAD (27 F/53 M) and 30 healthy volunteers (10 F/20 M), the following parameters were measured: Concentrations of fibrinogen, tissue-Plasminogen Activator (t-PA Ag), Plasminogen Activator Inhibitor-1 (PAI-1 Ag), D-dimer, and platelet (PLT) count. Results: In the blood of patients with PAD and concomitant DM 2 significantly higher concentrations of fibrinogen were found in comparison with patients with PAD and without diabetes (p = 0.044). No significant impact was observed in individuals with atherosclerotic complications (manifested by coronary artery disease, atherosclerosis of cerebral arteries) and selected cardiovascular risk factors (smoking, LDL and triglyceride concentrations, BP values) on the levels of t-PA, PAI-1, D-dimer, and PLT count. It was found that t-PA Ag and PAI-1 Ag values tended to rise along with a BMI increase in the subgroups of subjects (with normal body mass, overweight, and obesity), but no statistically significant differences were observed. However, two significant positive correlations were reported between t-PA Ag and BMI, as well as between PAI-1 Ag and BMI. Conclusions: Type 2 diabetes in peripheral arterial disease affects the concentration of fibrinogen causing its increase, which is connected with the inflammation and prothrombotic process in the course of both conditions. The concurrence of atherosclerosis of coronary or cerebral arteries, smoking, LDL and TG concentrations, and BP value do not have a significant impact on the levels of analyzed fibrinolysis parameters. A positive correlation between BMI and t-PA Ag and PAI-1 Ag concentrations needs to be supported in further studies on a larger number of overweight and obese patients.

Tissue factor levels and the fibrinolytic system in thin and thick intraluminal thrombus and underlying walls of abdominal aortic aneurysms.

BACKGROUND: The hemostatic system cooperates with proteolytic degradation in processes allowing abdominal aortic aneurysm (AAA) formation. In previous studies, it has been suggested that aneurysm rupture depends on intraluminal thrombus (ILT) thickness, which varies across each individual aneurysm. We hypothesized that hemostatic components differentially accumulate in AAA tissue in relation to ILT thickness. Thick (A1) and thin (B1) segments of ILTs and aneurysm wall sections A (adjacent to A1) and B (adjacent to B1) from one aneurysm sac were taken from 35 patients undergoing elective repair. METHODS: Factor levels were measured using enzyme-linked immunosorbent assay of protein extract. RESULTS: Tissue factor (TF) activities were significantly higher in thinner segments of AAA (B1 vs A1, P = .003; B vs A, P < .001; B vs A1, P < .001; B vs B1, P = .001). Significantly higher tissue plasminogen activator was found in thick thrombus-covered wall segments (A) than in B, A1, and B1 (P = .015, P < .001, and P < .001, respectively). Plasminogen concentrations were highest in ILT. Concentrations of α2-antiplasmin in thin ILT adjacent walls (B) were higher compared with wall (A) adjacent to thick ILT (P = .021) and thick ILT (A1; P < .001). Significant correlations between levels of different factors were mostly found in thick ILT (A1). However, no correlations were found at B sites, except for a correlation between plasmin and TF activities (r = 0.55; P = .004). CONCLUSIONS: These results suggest that higher TF activities are present in thinner AAA regions. These parameters and local fibrinolysis may be part of the processes leading to destruction of the aneurysm wall.

The effect of low-level laser therapy as an adjunct to non-surgical periodontal treatment on gingival crevicular fluid levels of transforming growth factor-beta 1, tissue plasminogen activator and plasminogen activator inhibitor 1 in smoking and non-smoking chronic periodontitis patients: A split-mouth, randomized control study.

BACKGROUND AND OBJECTIVE: This study aimed to investigate the effects of low-level laser therapy (LLLT) as an adjunct to scaling and root planing (SRP) on smoking and non-smoking patients with chronic periodontitis. MATERIAL AND METHODS: The study was conducted using a split-mouth design with 30 patients with chronic periodontitis (15 smokers, 15 non-smokers) and 30 healthy individuals matched for age, sex and smoking status as controls. Groups were constituted as follows: Cp+SRP+Sham: non-smokers with chronic periodontitis treated with SRP; Cp+SRP+LLLT: non-smokers with chronic periodontitis treated with SRP+LLLT; SCp+SRP+Sham: smokers with chronic periodontitis treated with SRP; SCp+SRP+LLLT: smokers with chronic periodontitis treated with SRP+LLLT; C: control group comprised of periodontally healthy non-smokers; SC: control group comprised of periodontally healthy smokers. LLLT was first applied on the same day as SRP and again on days 2 and 7 after SRP treatment. Clinical parameters were recorded before non-surgical periodontal treatment (baseline) and on day 30. Gingival crevicular fluid samples were collected before periodontal treatment (baseline) and during follow-up visits on days 7, 14 and 30. Gingival crevicular fluid transforming growth factor (TGF)-ß1, tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) levels were measured using enzyme-linked immunosorbent assay. RESULTS: All clinical parameters showed significant reductions between baseline and day 30 following SRP treatment in both the LLLT and sham groups (P<.001). No significant differences were observed between the LLLT and sham groups of either the smokers or non-smokers (P>.05). Gingival crevicular fluid PAI-1 levels decreased significantly in the SCp+SRP+sham and SCp+SRP+LLLT groups (P<.05), and gingival crevicular fluid tPA levels decreased significantly in the Cp+SRP+sham, Cp+SRP+LLLT and SCp+SRP+LLLT groups (P<.05). Gingival crevicular fluid TGF-ß1 levels decreased significantly in all treatment groups (P<.05). Although no significant differences were found between the gingival crevicular fluid PAI-1, tPA and TGF-ß1 levels of the LLLT versus sham groups (P>.05) at any of the time points measured, both LLLT groups showed significant reductions in tPA/PAI-1 ratios over time. CONCLUSION: Within the limits of this study, LLLT may be understood to play a role in the modulation of periodontal tissue tPA and PAI-1 gingival crevicular fluid levels, particularly in smoking patients with chronic periodontitis, and may thus be recommended as an adjunct to non-surgical periodontal treatment.

Host biomarkers detected in saliva show promise as markers for the diagnosis of pulmonary tuberculosis disease and monitoring of the response to tuberculosis treatment.

BACKGROUND: There is an urgent need for new tools for the rapid diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers maybe useful, especially if based on easily available samples. We investigated host biomarkers detected in saliva samples from individuals with suspected pulmonary TB disease, as tools for the diagnosis of TB disease and monitoring of the response to treatment. METHODS: We collected saliva samples from 104 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 33 host markers in stored saliva samples using a multiplex cytokine platform. Using a combination of clinical, radiological and laboratory results and a pre-established diagnostic algorithm, participants were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potentials of individual analytes were analysed by the receiver operator characteristics curve approach while the predictive abilities of combinations of analytes for TB disease were analysed by general discriminant analysis, with leave-one-out cross validation. RESULTS: Of the 104 individuals enrolled, 32 were pulmonary TB cases. There were significant differences in the levels of 10 of the markers investigated between the patients with TB disease and those with ORDs. However, the optimal diagnostic biosignature was a seven-marker combination of salivary CRP, ferritin, serum amyloid P, MCP-1, alpha-2-macroglobulin, fibrinogen and tissue plasminogen activator. This biosignature diagnosed TB disease with a sensitivity of 78.1% (95% CI, 59.6-90.1%) and specificity of 83.3% (95% CI, 72.3-90.7%) after leave-one-out cross validation. When compared to baseline levels, the concentrations of 9 markers including granzyme A, MCP-1, IL-1ß, IL-9, IL-10, IL-15, MIP-1ß, ferritin and serum amyloid A changed significantly by months 2 or 6 after initiation of TB treatment, thereby indicating that they might be useful in monitoring the response to TB treatment. CONCLUSION: We have identified candidate biomarkers in saliva, which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. These results require further validation in larger studies.

Keratinocyte Growth Factor Combined with a Sodium Hyaluronate Gel Inhibits Postoperative Intra-Abdominal Adhesions.

Postoperative intra-abdominal adhesion is a very common complication after abdominal surgery. One clinical problem that remains to be solved is to identify an ideal strategy to prevent abdominal adhesions. Keratinocyte growth factor (KGF) has been proven to improve the proliferation of mesothelial cells, which may enhance fibrinolytic activity to suppress postoperative adhesions. This study investigated whether the combined administration of KGF and a sodium hyaluronate (HA) gel can prevent intra-abdominal adhesions by improving the orderly repair of the peritoneal mesothelial cells. The possible prevention mechanism was also explored. The cecum wall and its opposite parietal peritoneum were abraded after laparotomy to induce intra-abdominal adhesion formation. Animals were randomly allocated to receive topical application of HA, KGF, KGF + HA, or normal saline (Control). On postoperative day 7, the adhesion score was assessed with a visual scoring system. Masson's trichrome staining, picrosirius red staining and hydroxyproline assays were used to assess the magnitude of adhesion and tissue fibrosis. Cytokeratin, a marker of the mesothelial cells, was detected by immunohistochemistry. The levels of tissue plasminogen activator (tPA), interleukin-6 (IL-6), and transforming growth factor ß1 (TGF-ß1) in the abdominal fluid were determined using enzyme-linked immunosorbent assays (ELISAs). Western blotting was performed to examine the expression of the TGF-ß1, fibrinogen and α-smooth muscle actin (α-SMA) proteins in the rat peritoneal adhesion tissue. The combined administration of KGF and HA significantly reduced intra-abdominal adhesion formation and fibrin deposition and improved the orderly repair of the peritoneal mesothelial cells in the rat model. Furthermore, the combined administration of KGF and HA significantly increased the tPA levels but reduced the levels of IL-6, tumor necrosis factor α (TNF-α) and TGF-ß1 in the abdominal fluid. The expression levels of TGF-ß1, fibrinogen and α-SMA protein and mRNA in the rat peritoneum or adhesion tissues were also down-regulated following the combined administration of KGF and HA. The combined administration of KGF and HA can significantly prevent postoperative intra-abdominal adhesion formation by maintaining the separation of the injured peritoneum and promoting mesothelial cell regeneration. The potential mechanism may be associated with rapid mesothelial cell repair in the injured peritoneum. This study suggests that combined administration of KGF and HA may be a promising pharmacotherapeutic strategy for preventing abdominal adhesions, which is worth further study, and has potential value in clinical applications.

Life-Course Partnership Status and Biomarkers in Midlife: Evidence From the 1958 British Birth Cohort.

OBJECTIVES: We examined the association between trajectories of partnership status over the life course and objectively measured health indicators in midlife. METHODS: We used data from 4 waves (1981, 1991, 2000, and 2002-2004) of the British National Child Development Study (NCDS), a prospective cohort study that includes all people born in Britain during 1 week in March 1958 (n = 18 558). RESULTS: After controlling for selection attributable to early-life and early-adulthood characteristics, we found that life-course trajectories of partnership status were associated with hemostatic and inflammatory markers, the prevalence of metabolic syndrome and respiratory function in midlife. Never marrying or cohabiting was negatively associated with health in midlife for both genders, but the effect was more pronounced in men. Women who had married in their late 20s or early 30s and remained married had the best health in midlife. Men and women in cohabiting unions had midlife health outcomes similar to those in formal marriages. CONCLUSIONS: Partnership status over the life course has a cumulative effect on a wide range of objectively measured health indicators in midlife.

Fibrinolytic activity of endothelial cells from different venous beds.

BACKGROUND: Little is known about the molecular biology of endothelial cells from different venous vascular beds. As a result, our treatment of deep vein thrombosis and pulmonary artery embolism remain identical. As an initial step in understanding venous thromboembolic disease in the trauma and surgical patients, this study sought to investigate the balance between coagulation and fibrinolysis in the pulmonary and deep venous vascular beds and how trauma might influence this balance. MATERIALS AND METHODS: Confluent human iliac vein endothelial cells (HIVECs) and human pulmonary artery endothelial cells (HPAECs), were cultured in the absence or presence of tumor necrosis factor (TNFα; 10 ng/mL) for 24 h. The expression of mediators of coagulation and fibrinolysis were determined by Western blot analysis, and plasminogen activator activity was determined by a fibrin clot degradation assay. RESULTS: After TNFα stimulation, there was decreased expression of endothelial protein C receptor and thrombomodulin in both HIVECs and HPAECs. TNFα stimulation increased urokinase plasminogen activator expression in both HIVECs and HPAECs. There was an increase in the expression of tissue plasminogen activator and plasminogen activator inhibitor-1 in response to TNFα in HPAECs, but not in HIVECs. There was significantly greater clot degradation in the presence of both the conditioned media and cell extracts from HIVECs, when compared with HPAECs. CONCLUSIONS: HPAECs and HIVECs react differently in terms of fibrinolytic potential when challenged with a cytokine associated with inflammation. These findings suggest that endothelial cells from distinct venous vascular beds may differentially regulate the fibrinolytic pathway.

Evaluation of gingival crevicular fluid levels of tissue plasminogen activator, plasminogen activator inhibitor 2, matrix metalloproteinase-3 and interleukin 1-ß in patients with different periodontal diseases.

OBJECTIVES: The purpose of this study was to evaluate the gingival crevicular fluid levels of interleukin-1beta (IL-1ß), matrix metalloproteinases-3 (MMP-3), tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) in patients with chronic periodontitis, aggressive periodontitis (AgP) and healthy individuals (controls). MATERIAL AND METHODS: Systemically healthy (21 chronic periodontitis, 23 AgP and 20 controls) subjects were included in this study. Plaque index, gingival index, probing pocket depth and clinical attachment level were recorded and gingival crevicular fluid samples were collected. Assays for IL-1ß, MMP-3, t-PA and PAI-2 levels in gingival crevicular fluid were carried out by an enzyme-linked immunosorbent assay. The one-sample Kolmogorov-Smirnov test, Mann-Whitney U test and Spearman correlation coefficient were used for data analyses. RESULTS: Gingival crevicular fluid levels of t-PA and IL-1ß were significantly higher in chronic periodontitis and AgP groups than in the control group (p < 0.001). MMP-3 levels in gingival crevicular fluid were detected as significantly higher in the chronic periodontitis and AgP groups compared with the control group (p < 0.05). The t-PA/PAI-2 rate of patients with chronic periodontitis and AgP were significantly higher than the control group (p < 0.05). The positive correlations were found among the PAI-2, t-PA, IL-1ß and MMP-3 levels in gingival crevicular fluid. The volume of the gingival crevicular fluid correlated with all of the clinical parameters (p < 0.001). There were positive correlations between the gingival crevicular fluid levels of PAI-2 and the probing pocket depth and between gingival crevicular fluid levels of PAI-2 and the clinical attachment level (p < 0.01). Similarly, significant correlations were found between t-PA levels and probing pocket depth and between t-PA levels and clinical attachment level measurements (p < 0.001). CONCLUSION: The present data showed that gingival crevicular fluid levels of IL-1 ß, MMP-3 and t-PA increased in periodontal disease regardless of the periodontitis type and played a part in tissue destruction.

Detection of targets and their mechanisms for early diagnosis of traumatic deep vein thrombosis.

The purpose of this investigation was to identify targets for the early diagnosis and predictors of deep venous thrombosis (DVT) and the role of these targets in the formation of venous thrombosis. A model of DVT was constructed in rats. Thromboses and venous walls were sampled for reverse transcription polymerase chain reaction study, and blood was sampled for enzyme-linked immunosorbent assay studies. Vein endothelial cells were cultured to observe the effects of interleukin (IL)-17 on the expression of tissue plasminogen activator (t-PA)/plasminogen activator inhibitor type 1 (PAI-1). IL-17 monoclonal antibody was used to study its effect on preventing the formation of DVT. One-hundred and twenty hours after the animal model was constructed, significant DVT started to form. Polymerase chain reaction tests showed that immediately after the model was created, the expression of IL-17 increased greatly, whereas the balance between t-PA and PAI-1 was disrupted just before DVT formed. The increase of serum IL-17 was positively related with the formation of DVT. Thus, the application of IL-17 monoclonal antibody could reduce the formation of DVT in rats. IL-17 might be a target for the early diagnosis of DVT and should be further studied to assess its clinical value.

Effects of addition of tissue-type plasminogen activator in in vitro fertilization medium on bovine embryo development and quality.

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.

[The correlation study of the expression of VEGF with t-PA and PAI expression in plasma and intraocular tissue in proliferative diabetic retinopathy].

OBJECTIVE: To evaluate the correlation between the expression of VEGF with t-PA and PAI expression in plasma aqueous humor and vitreous of proliferative diabetic retinopathy (PDR). METHODS: It was an experimental study. The concentrations of VEGF t-PA and PAI in plasma, aqueous humor and vitreous body in PDR and normal group were detected by ELISA. RESULTS: The levels of VEGF (53.37 ng/L) and PAI (8.00 µg/L) in the plasma of PDR patients were higher than control group, and there was significant statistically difference between them (Z = -3.437, -5.816; P < 0.01). The levels of t-PA (24.32 µg/L) in plasma of PDR patients and control group was no statistically difference between them (Z = -1.715, P = 0.086). The levels of VEGF (335.00 ng/L) t-PA (5.70 µg/L) and PAI (0.63 µg/L) in the aqueous humor of PDR patients were higher than control group, and there was statistically difference between them (Z = -4.805, -1.967, -4.018; P < 0.05). The levels of VEGF (691.69 ng/L) t-PA (13.05 µg/L) and PAI (4.01 µg/L) in the vitreous of PDR patients were higher than control group , and there was statistically difference between them (Z = -2.807, -2.642, -2.230;P < 0.05). Compare the plasma aqueous humor and vitreous of PDR patients. The levels of VEGF is: vitreous>aqueous humor>plasma. The levels of t-PA is: plasma>vitreous>aqueous humor. The levels of PAI is: plasma and vitreous>aqueous humor. The expression of VEGF was highly correlated with t-PA and PAI in plasma aqueous humor and vitreous of PDR (P < 0.05). CONCLUSIONS: VEGF PAI in plasma, VEGF t-PA PAI in aqueous humor and vitreous were increases. The expression of VEGF was positive correlated with t-PA and PAI in plasma aqueous humor and vitreous of PDR.

Tissue-type plasminogen activator and plasminogen embedded in casein rule its degradation under physiological situations: manipulation with casein hydrolysate.

The aims of this study were to test the assumption that tissue-type plasminogen activator (t-PA) and plasminogen (PG) are closely associated with the casein micelle and form a functional complex that rules casein degradation. This assumption was essentially verified for bovine milk under conditions wherein the plasmin system was activated by treatment with casein hydrolysate. It was also shown that urokinase-type PA (u-PA), the second type of plasminogen activator present in milk, was not involved in casein degradation. In agreement with previous studies, we show that treatment with casein hydrolysate precipitously reduced mammary secretion, disrupted the tight junction integrity (increase in Na+ and decrease in K+ concentrations), induced hydrolysis of casein, and activated various elements of the innate and acquired immune system. In the present study, we have identified t-PA as the principal PA, which is responsible for the conversion of PG to plasmin. It was found that t-PA and plasminogen are present in freshly secreted milk (less than 10 min from its secretion), suggesting that they are secreted as a complex by the mammary gland epithelial cells. Further research is needed to provide the direct evidence to verify this concept.

Profibrinolytic, antithrombotic, and antiinflammatory effects of an insulin-sensitizing strategy in patients in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial.

BACKGROUND: Effects were compared in patients in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial of 2 mechanistically different strategies for treatment of hyperglycemia, insulin-sensitizing and insulin-providing strategies, on biomarker profiles reflecting the balance between fibrinolysis and thrombosis and the intensity of inflammation implicated in diabetic vasculopathy. METHODS AND RESULTS: A total of 2368 patients with type 2 diabetes mellitus and clinically stable, angiographically documented coronary artery disease were randomized to treatment with 1 of the 2 strategies and followed for an average of 5 years. Plasminogen activator inhibitor type 1 antigen and activity, tissue plasminogen activator antigen, fibrinogen, D-dimer, C-reactive protein, insulin, and hemoglobin A(1c) were assayed in blood samples acquired at baseline and at 12 regular intervals throughout the follow-up interval. Higher baseline D-dimer, fibrinogen, and C-reactive protein portended a poor prognosis in patients in both groups. In contrast to the insulin-providing strategy, the insulin-sensitizing strategy led to (1) lower plasma insulin; (2) lower plasminogen activator inhibitor type 1 antigen and activity and lower tissue plasminogen activator antigen (known to track with plasminogen activator inhibitor type 1); and (3) lower C-reactive protein and fibrinogen at all intervals after baseline (P<0.001 for each). CONCLUSIONS: The insulin-sensitizing treatment strategy led to changes in biomarker profiles indicative of decreased insulin resistance, an altered balance between thrombosis and fibrinolysis favoring fibrinolysis, and diminished intensity of the systemic inflammatory state, factors that have been associated with cardiovascular risk. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT00006305.

Influenza A(H1N1)pdm09 Virus Alters Expression of Endothelial Factors in Pulmonary Vascular Endothelium in Rats.

Influenza virus infection may cause endothelial activation and dysfunction. However, it is still not known to what extent the influenza virus can dysregulate the expression of various endothelial proteins. The aim of the study is to identify the level of expression of endothelial nitric oxide synthase (eNOS), plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (tPA) in the pulmonary vascular endothelium, as well as the concentration of PAI-1 and tPA in the blood plasma in Wistar rats. Animals were intranasally infected with rat-adapted influenza A(H1N1)pdm09 virus. The expression of eNOS, PAI-1 and tPA in the pulmonary vascular endothelium was determined by immunohistochemistry; the concentration of PAI-1 and tPA was analyzed by ELISA at 24 and 96 h post infection (hpi). Thus, the expression of eNOS in the pulmonary vascular endothelium decreased by 1.9-fold at 24 hpi and increased by 2-fold at 96 hpi. The expression of PAI-1 in the pulmonary vascular endothelium increased by 5.23-fold and 6.54-fold at 24 and 96 hpi, respectively. The concentration of PAI-1 in the blood plasma of the rats decreased by 3.84-fold at 96 hpi, but not at 24 hpi. The expression of tPA in the pulmonary vascular endothelium was increased 2.2-fold at 96 hpi. The obtained data indicate the development of endothelial dysfunction that is characterized by the dysregulation of endothelial protein expression in non-lethal and clinically non-severe experimental influenza virus infection.