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Background: Sepsis is a systemic inflammatory response syndrome, associated with high risk of acute kidney injury (AKI) and in-hospital mortality. Thymosin beta-4 (Tβ4) is an actin-sequestering protein that can prevent inflammation in several tissues. Thus, we studied the role of Tβ4 in sepsis., Methods: The Tβ4 concentrations were prospectively measured in 191 patients within 6 h of the intensive care units (ICU) admission with diagnosis of sepsis. The cohort was divided into Tβ4 concentration tertiles: 1.19-7.11 ng/ml (n = 64), 7.12-11.01 ng/ml (n = 64), and 11.02-28.10 ng/ml (n = 63)., Results: Of 191 patients, 92 patients developed AKI, 24 of whom received continuous renal replacement therapy (CRRT), 29 patients died within 7 days, and 53 patients died within 28 days. Lower Tβ4 stages were correlated with poor prognosis, including AKI(odds ratio [OR], 2.102 per stage lower; 95% confidence interval [CI], 1.448 to 3.050; P < 0.001), CRRT(OR, 2.346 per stage lower; 95% CI, 1.287 to 4.276; P = 0.005), 7-day mortality(OR, 1.755 per stage lower; 95% CI, 1.050 to 2.935; P = 0.032), and 28-day mortality(OR, 1.821 per stage lower; 95% CI, 1.209 to 2.743; P = 0.004). Kaplan-Meier analysis also demonstrated that patients with lower Tβ4 stages had a high risk of AKI and death. In addition, the area under the curve (AUC) of Tβ4 for predicting AKI, CRRT, 7-day mortality, and 28-day mortality were, respectively, 0.702 (95% CI 0.628-0.776), 0.717 (95% CI 0.592-0.842), 0.694 (95% CI 0.579-0.808), and 0.682 (95% CI 0.598-0.767)., Conclusions: Lower Tβ4 stages are associated with higher odds of poor prognosis in ICU patients with sepsis., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: We previously demonstrated that plasma levels of F-actin and Thymosin Beta 4 differs among patients with septic shock, non-infectious systemic inflammatory syndrome and healthy controls and may serve as biomarkers for the diagnosis of sepsis. The current study aims to determine if these proteins are associated with or predictive of illness severity in patients at risk for sepsis in the Emergency Department (ED). Methods: Prospective, biomarker study enrolling patients (>18 years) who met the Shock Precautions on Triage Sepsis rule placing them at-risk for sepsis. Results: In this study of 203 ED patients, F-actin plasma levels had a linear trend of increase when the quick Sequential Organ Failure Assessment (qSOFA) score increased. F-actin was also increased in patients who were admitted to the Intensive Care Unit (ICU) from the ED, and in those with positive urine cultures. Thymosin Beta 4 was not associated with or predictive of any significant outcome measures. Conclusion: Increased levels of plasma F-actin measured in the ED were associated with incremental illness severity as measured by the qSOFA score and need for ICU admission. F-actin may have utility in risk stratification of undifferentiated patients in the ED presenting with signs and symptoms of sepsis.

Congenital heart disease is one of the largest class of birth defects. Eight subjects with ventricular septal defect (VSD, a kind of congenital heart disease) and 11 health children were enrolled in tandem mass tags label-based quantitative proteomic analysis to compare plasma proteins differentially abundance. A total of 66 proteins were significantly upregulated or downregulated in VSD patients compared with healthy children. These proteins were involved in pathways linked to platelet activation, fructose and mannose metabolism, complement and coagulation cascades, glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and carbon metabolism. The amount of ten proteins changed significantly (p < 0.05) in newly recruited 30 VSD compared with 15 control children, which were validated by ELISA. The areas under the receiver operating characteristic curve values of fructose-bisphosphate aldolase B (ALDOB) and thymosin beta-4 (Tβ4) were higher than those of other candidate proteins. ALDOB and Tβ4 might be potential biomarkers applied for identifying VSD in the further works., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and objectives : Ischemia-reperfusion (IR) caused by infrarenal abdominal aorta cross-clamping is an important factor in the development of ischemia-reperfusion injury in various distant organs. Materials and Methods: We investigated potential antioxidant/anti-inflammatory effects of thymosin beta 4 (Tβ4) in a rat model of abdominal aortic surgery-induced IR. Tβ4 (10 mg/kg, intravenous (i.v.)) was administered to rats with IR (90-min ischemia, 180-min reperfusion) at two different periods. One group received Tβ4 1 h before ischemia, and the other received 15 min before the reperfusion period. Results: Results were compared to control and non-Tβ4-treated rats with IR. Serum, bronchoalveolar lavage fluid and lung tissue levels of oxidant parameters were higher, while antioxidant levels were lower in the IR group compared to control. IR also increased inflammatory cytokine levels. Tβ4 reverted these parameters in both Tβ4-treated groups compared to the untreated IR group. Conclusions: Since there is no statistical difference between the prescribed results of both Tβ4-treated groups, our study demonstrates that Tβ4 reduced lung oxidative stress and inflammation following IR and prevented lung tissue injury regardless of timing of administration.

Background and Aims: non-alcoholic fatty liver disease (NAFLD) is the most common type of chronic liver injury worldwide. Some studies have shown that thymosin beta4 (Tβ4) is closely related to liver diseases. Nevertheless, only a few published studies have reported the relationship between Tβ4 and NAFLD. The purpose of this study was to evaluate the levels of Tβ4 in patients with NAFLD compared with controls and to validate their relationship in a larger cohort., Patients and Methods: a total of 76 NAFLD patients and 130 healthy controls were included in the study. Serum levels of Tβ4, IL-6 and adiponectin were determined by ELISA. Serum glucose, insulin and lipids, as well as liver function were measured. Multivariate statistical analyses were performed via logistic regression modelling to determine the predictors with a significant relevance to NAFLD. The association between serum Tβ4 and study variables was tested using correlation coefficients calculations., Results: serum Tβ4 content was 3.20 ± 0.98 mg/l in NAFLD patients (n = 76) and 5.53 ± 1.24 mg/l in healthy controls (n = 130); the difference between the two groups was statistically significant (p = 0.000). Multivariate logistic regression analysis identified Tβ4 (OR = 0.343, 95% CI 0.240-0.491, p < 0.001), LDL (OR = 1.019, 95% CI 1.007-1.030, p = 0.001), ALT (OR = 1.021, 95% CI 1.001-1.041, p = 0.040) and IL-6 (OR = 1.443, 95% CI 1.079-1.929, p = 0.013) as independent predictors of NAFLD diagnosis. Serum Tβ4 levels had a significant negative correlation with total cholesterol, TG, AST, GGT and IL-6 (p < 0.05 for all) and the correlation coefficient values were -0.163, -0.253, -0.143, -0.245 and -0.155, respectively. Serum Tβ4 levels were positively correlated with serum adiponectin levels, with a correlation coefficient value of 0.143., Conclusion: serum Tβ4 may play a defensive role in the development of NAFLD. Further studies are needed to confirm the role of Tβ4 in NAFLD.

In the hospital, blood samples are collected to monitor patients' health states, and thus various protein-based clinical methods have been developed. However, some proteins are found to change in abundances during the process of blood collection and storage. In order to account such pre-analytical effects, we performed liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) on 15 selected proteins in plasma samples prepared by varying storage time and temperature of whole blood prior to plasma isolation. Two cytosolic proteins, profilin-1 (PFN1) and thymosin beta-4 (TMSB4X), were absolutely quantified using 15 N-labeled recombinant proteins spiked externally. The other 13 proteins were quantified in a relative way compared with the two reference proteins. Triplicated LC-MRM-MS measurements showed that the median CV of MRM peak areas was 5.7%. The amounts of PFN1 and TMSB4X increased rapidly depending on the storage time between blood collection and plasma preparation. It indicates the leakage of cellular components into the plasma fraction. Relative quantification further revealed that five proteins including PFN1, S10A8, S10A9, S10A11, and TMSB4X showed significant difference (P < 0.05). We further monitored PFN1 and TMSB4X on 40 samples collected for protein diagnostics under a typical clinical study condition. Compared with the plasma samples prepared within a day, the level of both PFN1 and TMSB4X increased in the plasma samples prepared from the blood collected the day before and kept overnight at 4°C (0.51 to 3.11 μg/mL for PFN1 and 0.98 to 5.36 μg/mL for TMSB4X in average). Our result suggests an effort of assuring plasma quality for accurate protein-based diagnosis or biomarker discovery and validation., (© 2018 John Wiley & Sons, Ltd.)

Introduction: Thymosin beta-4 (TB4) is an endogenous peptide with protective and regenerative effects in models of cellular and organ injury. TB4 is increasingly measured as a potential plasma or serum biomarker in human cardiovascular, liver, infectious, and autoimmune disease., Areas Covered: The focus of this review is the quantification of TB4 in clinical cohort studies and whether reported TB4 concentrations differ with respect to method of sample preparation. We survey current literature for studies measuring TB4 in human serum or plasma and compare reported concentrations in healthy controls., Expert Opinion: We find substantial intra- and inter- study variability in healthy controls, and a lack of protocol standardization. We further highlight three factors that may confound TB4 clinical measurements and should be considered in future study design: 1) residual platelets remaining in suspension after centrifugation, 2) TB4 release following ex vivo platelet activation, and 3) specificity of assays towards posttranslational modifications. Accordingly, we put forth our recommendations to minimize residual and activated platelets during sample collection, and to cross-validate TB4 measurements using both antibody-based and mass spectrometry-based methods.

Objective: The aim of the study was to determine whether serum thymosin beta4 (Tβ4) can be a useful noninvasive biomarker to differentiate between nonalcoholic steatohepatitis (NASH) and nonalcoholic fatty liver (NAFL)., Methods: The study included 24 NAFL patients and 21 NASH patients. The levels of Tβ4, 8-hydroxydeoxyguanosine acid (8-OhdG), liver function parameters, blood lipid, and glucose were detected in the venous blood of all patients. The NAFLD histological activity score (NAS) was examined in biopsy specimens from all patients. Statistical analysis was performed in order to find differences between the two abovementioned groups. In addition, receiver operator characteristic (ROC) analyses for alanine aminotransferase (ALT) and Tβ4 levels were performed in NAFL and NASH patients and the cut-off value was determined. Associations between the variables were tested using correlation coefficient calculations. Statistical significance was set at a p value of < 0.05., Results: Serum Tβ4 content was 5.12 ± 1.87 mg/l in the NAFL group and 2.98 ± 1.35 mg/l in the NASH group (p < 0.001). Serum Tβ4 content and NAS, histological features of hepatic steatosis, lobular inflammation and ballooning, ALT, glucose and 8-OhdG levels were negatively correlated (p < 0.05 for all) in the NASH group. The correlation coefficient values were -0.530, -0.562, -0.574, -0.438, -0.446, -0.426 and -0.563, respectively. On the basis of ROC analysis, the best predictive Tβ4 cut-off value for detecting NASH was 3.94 mg/l (85.7% sensitivity and 79.2% specificity, which were higher than those of ALT)., Conclusion: Serum Tβ4 level can be used as a biomarker for the diagnosis of NASH and was negatively correlated with the oxidation state of the liver.

Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.

Background: Thymosin beta-4 (TB4) is an X-linked gene product with cardioprotective properties. Little is known about plasma concentration of TB4 in heart failure (HF), and its relationship with other cardiovascular biomarkers. We sought to evaluate circulating TB4 in HF patients with preserved (HFpEF) or reduced (HFrEF) ejection fraction compared to non-HF controls., Methods and Results: TB4 was measured using a liquid chromatography and mass spectrometry assay in age- and sex-matched HFpEF (n=219), HFrEF (n=219) patients, and controls (n=219) from a prospective nationwide study. Additionally, a 92-marker multiplex proximity extension assay was measured to identify biomarker covariates. Compared with controls, plasma TB4 was elevated in HFpEF (985 [421-1723] ng/mL versus 1401 [720-2379] ng/mL, P <0.001), but not in HFrEF (1106 [556-1955] ng/mL, P =0.642). Stratifying by sex, only women (1623 [1040-2625] ng/mL versus 942 [386-1891] ng/mL, P <0.001), but not men (1238.5 [586-1967] ng/mL versus 1004 [451-1538] ng/mL, P =1.0), had significantly elevated TB4 in the setting of HFpEF. Adjusted for New York Heart Association class, N-terminal pro B-type natriuretic peptide, age, and myocardial infarction, hazard ratio to all-cause mortality is significantly higher in women with elevated TB4 (1.668, P =0.036), but not in men (0.791, P =0.456) with HF. TB4 is strongly correlated with a cluster of 7 markers from the proximity extension assay panel, which are either X-linked, regulated by sex hormones, or involved with NF-κB signaling., Conclusions: We show that plasma TB4 is elevated in women with HFpEF and has prognostic information. Because TB4 can preserve EF in animal studies of cardiac injury, the relation of endogenous, circulating TB4 to X chromosome biology and differential outcomes in female heart disease warrants further study., (© 2017 The Authors and AstraZeneca. Published on behalf of the American Heart Association, Inc., by Wiley.)

Background: Thymosin beta 10 (TMSB10) has been demonstrated to be involved in the malignant process of many cancers. The purpose of this study was to determine the biological roles and clinical significance of TMSB10 in breast cancer and to identify whether TMSB10 might be used as a serum marker for the diagnosis of breast cancer., Methods: TMSB10 expression was evaluated by immunohistochemical analysis (IHC) of 253 breast tumors and ELISA of serum from 80 patients with breast cancer. Statistical analysis was performed to explore the correlation between TMSB10 expression and clinicopathological features in breast cancer. Univariate and multivariate Cox regression analysis were performed to examine the association between TMSB10 expression and overall survival and metastatic status. In vitro and in vivo assays were performed to assess the biological roles of TMSB10 in breast cancer. Western blotting and luciferase assays were examined to identify the underlying pathway involved in the tumor-promoting role of TMSB10., Results: We found TMSB10 was upregulated in breast cancer cells and tissues. Univariate and multivariate analysis demonstrated that high TMSB10 expression significantly correlated with clinicopathological features, poor prognosis and distant metastases in patients with breast cancer. Overexpression of TMSB10 promotes, while silencing of TMSB10 inhibits, proliferation, invasion and migration of breast cancer cells in vitro and in vivo. Our results further reveal that TMSB10 promotes the proliferation, invasion and migration of breast cancer cells via AKT/FOXO signaling, which is antagonized by the AKT kinase inhibitor perifosine. Importantly, the expression of TMSB10 is significantly elevated in the serum of patients with breast cancer and is positively associated with clinical stages of breast cancer., Conclusion: TMSB10 may hold promise as a minimally invasive serum cancer biomarker for the diagnosis of breast cancer and a potential therapeutic target which will facilitate the development of a novel therapeutic strategy against breast cancer.

Thymosin alpha 1 (Tα1) is a powerful modulator of immunity and inflammation. Despite years of studies, there are a few reports evaluating serum Tα1 in health and disease. We studied a cohort of healthy individuals in comparison with patients affected by chronic inflammatory autoimmune diseases. Sera from 120 blood donors (healthy controls, HC), 120 patients with psoriatic arthritis (PsA), 40 with rheumatoid arthritis (RA) and 40 with systemic lupus erythematosus (SLE), attending the Transfusion Medicine or the Rheumatology Clinic at the Policlinico Tor Vergata, Rome, Italy, were tested for Tα1 content by means of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Data were analysed in relation to demographic and clinical characteristics of patients and controls. A gender difference was found in the HC group, where females had lower serum Tα1 levels than males (P < 0·0001). Patients had lower serum Tα1 levels than HC (P < 0·0001), the lowest were observed in PsA group (P < 0·0001 versus all the other groups). Among all patients, those who at the time of blood collection were taking disease-modifying anti-rheumatic drugs (DMARD) plus steroids had significantly higher Tα1 levels than those taking DMARD alone (P = 0·044) or no treatment (P < 0·0001), but not of those taking steroids alone (P = 0·280). However, whichever type of treatment was taken by the patients, serum Tα1 was still significantly lower than in HC and there was no treatment-related difference in PsA group. Further prospective studies are necessary to confirm and deepen these observations. They might improve our understanding on the regulatory role of Tα1 in health and disease and increase our knowledge of the pathogenesis of chronic inflammatory autoimmune diseases., (© 2016 British Society for Immunology.)

Objective: Inflammation is a potential mechanism of obstructive sleep apnea syndrome (OSAS). Thymosin β4, a member of thymic protein family, exhibits an anti-inflammatory effect. We determine to investigate whether serum thymosin β4 concentrations is correlated with the occurrence and disease severity of OSAS., Methods: Serum thymosin β4 concentrations were examined in a cross-sectional population including 158 patients with OSAS and 94 healthy subjects., Results: Elevated serum thymosin β4 concentrations were found in OSAS patients than the controls. Multivariable logistic regression analysis indicated a significant association between serum thymosin β4 concentrations and OSAS development. Severe OSAS patients showed increased serum thymosin β4 concentrations compared with mild and moderate patients. Spearman correlation analysis suggested that serum thymosin β4 concentrations were correlated with the severity of OSAS. Simple linear regression analyses showed that serum thymosin β4 in OSAS patients was correlated with homeostasis model assessment of insulin resistance, apnea hypopnea index, disease severity, and osteoarthritis development. Then multiple stepwise regression analysis showed that only disease severity remained to be associated with serum thymosin β4., Conclusions: Serum thymosin β4 concentrations were correlated with the occurrence and severity of OSAS., (© 2016 Wiley Periodicals, Inc.)

Objective: To compare plasma levels of F-actin, G-actin and thymosin beta 4 (TB4) in humans with septic shock, noninfectious systemic inflammatory response syndrome (SIRS) and healthy controls., Results: F-actin was significantly elevated in septic shock as compared with noninfectious SIRS and healthy controls. G-actin levels were greatest in the noninfectious SIRS group but significantly elevated in septic shock as compared with healthy controls. TB4 was not detectable in the septic shock or noninfectious SIRS group above the assay's lowest detection range (78 ng/ml)., Conclusions: F-actin is significantly elevated in patients with septic shock as compared with noninfectious SIRS. F-actin and the F:G-actin ratio are potential biomarkers for the diagnosis of septic shock.

Aim: Tβ4 is an integral factor in repair of myocardium in animal models. To investigate whether Tβ4 is important in human cardiac disease and has a role in mediating the beneficial cardiac effects of bone-marrow-derived stem cell (BMSC) therapy, we measured serial plasma Tβ4 levels in patients enrolled on the REGENERATE-IHD cell therapy trial., Patients & Methods: Plasma Tβ4 concentrations were measured in 13 patients who received BMSCs and 14 controls., Results: There was a significant increase in plasma Tβ4 in the BMSC group 24 h after intracardiac injection. Increases in Tβ4 levels were associated with improvement in New York Heart Association symptom class., Conclusion: This exploratory study highlights the need for further study of Tβ4 in human cardiovascular disease.

Background and Aim: In the previous study, we found serum thymosin β4 (Tβ4) levels were associated with mortality in liver failure patients. In this study, we try to evaluate the prognostic value of Tβ4 in acute-on-chronic liver failure (AoCLF) patients by comparing with the Child-Pugh (CTP) and Model for End-Stage Liver Disease (MELD) scores., Methods: Serum Tβ4 levels were measured by enzyme-linked immunosorbent assay (ELISA), and the CTP and MELD scores were calculated for each patient., Results: Serum Tβ4 levels of AoCLF patients [0.4120 (0.2447-0.7492)μg/mL] were lower than healthy controls [9.2710 (5.1660-13.2485)μg/mL] (P<0.001). AoCLF patients were divided into survival and death group. Compared to survivors, lower Tβ4 concentrations, higher CTP and MELD scores (P<0.001, respectively) were observed in AoCLF patients who died. There were negative correlations between Tβ4 levels and CTP scores (P<0.001), MELD scores (P<0.001). A CTP score of 11.5, a MELD score of 21.63 and a Tβ4 concentration of 0.3840μg/mL were identified as the cut-off values for the stratification of AoCLF patients. MELD≥21.63 combined with Tβ4<0.3840μg/mL can more exactly discriminate between the patients who would survive and die., Conclusions: Serum Tβ4 concentration has appreciable value to evaluate the short-term prognosis of AoCLF patients, although Tβ4 is not superior to MELD. The combination of Tβ4 and MELD scores are more effective in assessing the prognosis of AoCLF patients., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Objectives: To determine the plasma levels of thymosin-α1 (TA1) and prothymosin-α (PTMA) proteins in renal cell carcinoma (RCC) or urothelial carcinoma (UC) patients, and explore the potential of these 2 molecules as biomarkers., Materials and Methods: Blood samples were taken from 50 consecutive patients with RCC, 97 with UC, and 55 with benign urologic diseases before surgery. Their clinical characteristics were obtained from medical record review. Plasma TA1 and PTMA levels were measured using enzyme-linked immunosorbent assay and their correlation with tumor grade, pathologic stage, and survival were explored., Results: Plasma TA1 levels were significantly lower in RCC patients than in UC or benign patients, particularly in UC of the renal pelvis patients (P < 0.0001). Plasma PTMA levels were also significantly lower in UC patients compared with RCC patients and benign patients (P < 0.05). Plasma TA1 levels inversely correlated with pathologic stage both in bladder cancer and RCC patients (P = 0.03 and 0.02, respectively). Both plasma TA1 and PTMA did not correlate with tumor grade. Plasma TA1 was a prognostic indicator for progression-free and disease-specific overall survival in bladder cancer patients (P = 0.008 and 0.04, respectively)., Conclusions: Plasma TA1 level may be a biomarker for differentiating between UC and RCC. It may also be a prognostic factor for disease progression and disease-specific survival in bladder cancer patients. These findings warrant more studies for validation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC., Design and Methods: The present work is a case-control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy., Results: A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1, respectively., Conclusions: The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b-c₁ complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers., (Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Apoptosis is characterized by a series of discrete biochemical events, among which is the truncation of the nuclear polypeptide prothymosin alpha (proTα) by activated caspase-3. This early apoptotic event results in the generation of a carboxy-terminal fragment of proTα, the immunoactive decapeptide proTα(100-109). We hypothesized that the detection of increased levels of proTα(100-109) in serum can be directly correlated with the induction of massive cell apoptosis, resulting from a severe bacterial infection. Thus, using high-affinity-purified polyclonal antibodies (Abs), raised in rabbits and a prototype antibody-capture system, we developed a highly sensitive and specific competitive ELISA for proTα(100-109). The sensitivity of the ELISA (0.1ng/mL to 10μg/mL) is acceptable for the quantification of the decapeptide in serum samples. To assess our initial hypothesis, we determined the concentration of proTα(100-109) in the sera of mice infected with the bacterium Streptococcus pyogenes over the course of the infection. We show that serum concentration of proTα(100-109) was marginal to undetectable before infection, increased over time and peaked at 72h postinfection. In silico analysis suggests that the Abs generated are unlikely to cross-react with any other unrelated mouse or bacterial protein. Further validation of our ELISA using serum samples from humans, infected with bacteria, may provide a useful tool to differentiate the causative agent of a potentially lethal septic infection., (© 2013.)

Objective: Thymosin β4, a member of a large family of thymic proteins, plays an important role in the process of articular cartilage degeneration which is a common cause of osteoarthritis (OA). This study aims to determine thymosin β4 levels in the serum and synovial fluid (SF) of patients with knee OA and analyze the correlation of thymosin β4 levels with the radiographic severity of OA., Methods: This study consisted of 216 patients with knee OA and 152 healthy controls. OA progression was classified based on Kellgren-Lawrence by evaluating x-ray changes observed in anteroposterior knee radiography. Thymosin β4 levels in the serum and SF were measured by enzyme-linked immunosorbent assay method., Results: The knee OA patients had higher levels of serum thymosin β4 than the healthy controls. Knee OA patients with KL grade 4 showed significantly elevated thymosin β4 levels in the serum and SF compared with those with KL grades 2 and 3. Knee OA patients with KL grade 3 had significantly higher SF levels of thymosin β4 than those with KL grade 2. Thymosin β4 levels in the serum and SF of knee OA patients were significantly correlated with disease severity according to KL grading criteria., Conclusion: The thymosin β4 levels in the serum and SF may serve as effective biomarkers for the severity of OA., (© 2013.)

A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment (17)LKKTETQ(23) is the active site within the protein thymosin β(4) responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography-mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The formulation TB-500 is suspected to be used as doping agent in sport. This work describes the detection and the identification of the N-terminal acetylated 17-23 fragment of human thymosin beta 4 (Ac-LKKTETQ) in TB-500 by means of high-performance liquid chromatography/high resolution mass spectrometry using an Orbitrap Exactive benchtop mass spectrometer. Ac-LKKTETQ was also synthesized by solid-phase peptide synthesis, and an analytical strategy for detection in plasma and urine by high-performance liquid chromatography/low resolution triple-quadrupole mass spectrometry was suggested.

The aim of this study was to evaluate whether thymosin β4 (Tβ4) levels are increased in the serum of rheumatoid arthritis (RA) patients, and if this increase is associated with RA disease activity and resistance to treatment. Blood samples from 40 patients with RA were collected at baseline and 6 months after starting treatment with disease-modifying antirheumatic drugs (DMARD) and/or tumor necrosis factor (TNF)-α blocker. Serum levels of Tβ4 were measured by ELISA. Tβ4 levels (mean ± standard deviation) in RA patients were significantly (approximately tenfold) higher than in healthy controls (577.4 ± 67.92 vs. 56.61 ± 5.72 ng/mL). Serum Tβ4 levels in patients with severe disease activity before therapy were slightly higher than in patients with moderate disease activity (662.4 ± 491.5 vs. 462.5 ± 305.3 ng/ml, P > 0.05). Tβ4 levels were significantly associated with disease activity according to the 28-joint Disease Activity Score. The mean Tβ4 level at baseline in the DMARD treatment group was significantly lower than in the DMARD + TNF-α blocker treatment group. Tβ4 levels were increased in the serum of patients with RA and were positively associated with disease activity. Levels of Tβ4 may also be relevant in determining or predicting resistance to RA treatment. Further studies are necessary to determine if Tβ4 is an appropriate therapeutic target for controlling inflammation associated with RA.

Objectives: Thymosin beta 4 (Tβ4) plays an essential role in cardiac vessel development and is currently being developed as a therapeutic agent for the treatment of coronary artery disease (CAD) in some experimental studies. Thus, we aimed to investigate the association of serum Tβ4 levels and collateral formation in patients presenting with severely stenotic CAD., Methods: Thirteen patients with poor collateral development and 16 age- and sex-matched patients with good collateral development who had ≥ 95% stenosis in at least one major coronary artery on coronary angiogram (CAG) were enrolled in the study. The Gensini score was calculated for each patient by using CAG results. Collateral development was classified according to the Cohen-Rentrop method. Serum Tβ4 levels were measured with enzyme-linked immune sorbent assay., Results: There were no statistically significant differences between the two groups in regard to clinical and laboratory characteristics of the patients except for Tβ4 levels. The Tβ4 levels in the well-collateralized study group were found to be significantly higher than those of the poorly collateralized study group and serum Tβ4 levels were positively correlated with the collateral development., Conclusions: Our findings suggest that serum Tβ4 levels are significantly associated with the collateral development in severe CAD.

Objectives: Thymosin beta4 (Tβ4) has been shown to have an important role in healing of damaged tissues and promoting cardiomyocyte survival in acute coronary syndromes. We evaluated endogenous Tβ4 levels in patients presenting with ST-elevation acute myocardial infarction (STEMI) before and after successful primary percutaneous coronary intervention (PCI)., Study Design: The study included 24 consecutive patients (7 females, 17 males; mean age 55.0±10.9 years) who underwent successful primary PCI for STEMI and 24 age- and sex-matched healthy controls (13 females, 11 males; mean age 57.5±11.7 years) with angiographically normal coronary arteries. To determine Tβ4 levels, blood samples were obtained from STEMI patients on admission and 48 hours after successful PCI, and from controls immediately after coronary angiography., Results: Compared to controls, baseline levels of high-density lipoprotein cholesterol (46.2±8.9 vs. 34.2±7.2 mg/dl, p<0.001) and Tβ4 (2.9±1.5 vs. 1.5±1.0 µg/ml, p<0.001) were significantly lower, and white blood cell counts (7.6±2.2 vs. 11.4±3.0 10³/µl, p<0.001) were significantly higher in the STEMI group. After 48 hours of PCI, the mean Tβ4 level increased significantly to 2.3±0.8 µg/ml (p<0.001) and became similar to that of the control group (p=0.068). There was a significant negative correlation between serum Tβ4 and white blood cell count (r=-0.347, p=0.016)., Conclusion: Considering the significant increase in serum Tβ4 levels following successful primary PCI in patients with STEMI, Tβ4 may prove to be a new marker in the assessment of reperfusion success in addition to those used currently.

Despite tremendous efforts in disclosing the pathophysiological and epidemiological factors associated with liver fibrogenesis, non-invasive diagnostic measures to estimate the clinical outcome and progression of liver fibrogenesis are presently limited. Therefore, there is a mandatory need for methodologies allowing the reasonable and reliable assessment of the severity and/or progression of hepatic fibrogenesis. We here performed proteomic serum profiling by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in 179 samples of patients chronically infected with hepatitis C virus and 195 control sera. Multidimensional analysis of spectra allowed the definition of algorithms capable to distinguish class-specific protein expression profiles in serum samples. Overall about 100 peaks could be detected per single spectrum. Different algorithms including protein peaks in the range of 2000 and 10,000 Da were generated after pre-fractionation on a weak cation exchange surface. A specificity of 93% with a sensitivity of 86% as mean of the test set results was found, respectively. The nature of three of these protein peaks that belonged to kininogen-1 and thymosin-β(4) was further analysed by tandem mass spectrometry (MS)/MS. We further found that kininogen-1 mRNA was significantly down-regulated in cirrhotic livers. We have identified kininogen-1 and thymosin-β(4) as potential new biomarkers for human chronic hepatitis C and conclude that serum profiling is a reliable technique to identify hepatitis-associated expression patterns. Based on the high throughput capability, the identified differential protein panel may serve as a diagnostic marker and warrants further validation in larger cohorts., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Purpose: To examine an association between thymosin β4 as potentially angioproliferative factor and proliferative diabetic retinopathy., Methods: The clinical study part included 62 patients with proliferative diabetic retinopathy (PDR) (study group) and 24 patients with non-diabetic pre-retinal membranes (control group). All patients underwent pars plana vitrectomy. We examined the thymosin β4 concentration in vitreous and plasma; and the expression of thymosin β4, glial fibrillary acidic protein (GFAP) and CD31 (PECAM-1 or Platelet Endothelial Cell Adhesion Molecule) and the levels of thymosin β4 mRNA and vascular endothelial growth factor (VEGF) mRNA in the excised membranes. The experimental study part consisted of 24 Sprague--Dawley rats with streptozotocin-induced diabetes mellitus and 24 age-matched control animals without diabetes. We determined the mRNA concentrations of thymosin β4, VEGF and GFAP in the rat retinas., Results: In the clinical study part, the vitreal and plasma thymosin β4 concentrations were significantly higher in the study group than control group (p =0.04 and p=0.01, respectively), and were significantly (p=0.028) correlated with each other. Co-expression of thymosin β4 and CD31 was observed in the diabetic fibrovascular membranes. Thymosin β4 mRNA and VEGF mRNA levels were significantly (p<0.01) higher in diabetic membranes than in non-diabetic membranes. In the experimental study part, the diabetic retinas showed co-localization of thymosin β4 and GFAP. The mRNA levels of thymosin β4, VEGF and GFAP were significantly (p<0.01) higher in diabetic rats than in control animals., Conclusions: Thymosin β4 was produced in intraocular fibrovascular membranes of patients with PDR and in rats with experimental diabetes mellitus. Thymosin β4 may play a role in diabetic retinal neovascularization., (© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.)

Background: Thymosin β4 was recently demonstrated to be a potential stimulator of angiogenesis and arteriogenesis in both in-vivo and in-vitro studies. Little is known about the relationship between serum thymosin β4 levels and the extent of coronary collaterals in patients with coronary artery disease., Hypothesis: Serum thymosin β4 independently predicts the formation of coronary collaterals in patients with coronary artery disease., Materials and Methods: A total of 190 consecutive patients who underwent coronary angiography were divided into two groups according to coronary angiograms and collateral formation: the poor collateral group consisting of 103 patients with at least more than or equal to one coronary stenosis of greater than or equal to 75% without visible collateral circulation, and the good collateral group consisting of 87 patients with at least more than or equal to one coronary stenosis of greater than or equal to 75% with well-developed collateral circulation. Collateral score was analyzed using the Cohen-Rentrop classification., Results: Serum thymosin β4 levels were 1024.50 ± 516.92 ng/ml and 1373.00 ± 1082.88 ng/ml for patients in the poor collateral group and the good collateral group, respectively. Serum thymosin β4 levels in the good collateral group were significantly higher than those in the poor collateral group (P<0.01). After adjustment in the multiple ordinal logistic regression model, serum thymosin β4 levels showed a strong independent association with the collateral Cohen-Rentrop score (χ(2)=4.45, odds ratio=1.00, 95% confidence interval: 1.000-1.002, P=0.035)., Conclusion: Elevated serum thymosin β4 level is independently associated with a significant enhancement in coronary collateralization and patients in the good collateral tend to have a higher thymosin β4 level.

Aim: To investigate whether serum thymosin beta4 can provide diagnostic or prognostic information in liver failure patients caused by chronic hepatitis B virus (HBV) infection., Methods: Serum thymosin beta4 levels were measured in 30 patients with acute-on-chronic liver failure (ACLF), 31 patients with chronic liver failure (CLF), 30 patients with compensated liver cirrhosis (CR) and 32 patients with chronic hepatitis B and 30 healthy controls. Serum thymosin beta4 levels were measured by enzyme-linked immunosorbent assay and Child-Pugh and model for end-stage liver disease (MELD) scores were calculated for each patient on admission., Results: Compared with healthy controls, serum thymosin beta4 levels in ACLF, CLF, CR and chronic hepatitis B patients were significantly lower, 6.5047 (4.7879-10.5314) microg/mL vs 0.4632 (0.2759-0.8768) microg/mL, 0.6981 (0.5209-1.2008) microg/mL, 1.8053 (0.8110-2.3397) microg/mL, 3.7803 (1.8570-6.4722) microg/mL, respectively (P < 0.001). The levels of thymosin beta4 in liver failure (ACLF or CLF) patients were markedly lower than that in CR (P < 0.001), and a difference was also found between CLF and ACLF patients (P = 0.038). In patients with chronic liver disease, there was a positive relationship between thymosin beta4 levels and albumin, choline esterase, and platelet (P < 0.001), and negative relationship with alanine aminotransferase (P = 0.020), aspartate aminotransferase, total bilirubin, international normalized ratio of prothrombin time, and Child-Pugh and MELD scores (P < 0.001). Of the 61 liver failure patients, the thymosin beta4 levels of non-survivors were significantly lower than that of survivors (P = 0.007). Receiver operating characteristics analysis identified a thymosin beta4 cutoff level of 0.5708 microg/mL for predicting poor prognosis in all liver failure patients. The serial thymosin beta4 values were observed in 13 liver failure inpatients. Lower initial values were observed in the death. While greater improvement in thymosin beta4 value was found in those who recovered from the disease., Conclusion: Serum thymosin beta4 can be used as an important potential predictor for liver failure caused by chronic HBV infection.

The beta-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin beta4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin beta4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that beta-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the beta-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the beta-thymosins, i.e., their modulating effect on the actin cytoskeleton.

beta-thymosins constitute a family of highly conserved 5-kDa polypeptides. Thymosin beta(4), the most abundant member of this family, is expressed in most mammalian cell types and is regarded as the main intracellular G-actin sequestering peptide. In addition to this important intracellular function several other activities have been attributed to this peptide. Thymosin beta(4) is released from human platelets and cross-linked to fibrin after activation of platelets with thrombin. While in most mammalian tissues thymosin beta(4) is accompanied by a second member of this peptide family, in human platelets only thymosin beta(4) is present. To elucidate if it is common to mammalian platelets that only one beta-thymosin is present, we analyzed platelets from several mammals for their beta-thymosin content. In human platelets only thymosin beta(4) could be detected, whereas in bovine platelets thymosin beta(9), which is normally the minor beta-thymosin in bovine tissues, was identified as the main beta-thymosin. In rabbit platelets, thymosin beta(4) is not simply replaced by the most homologous thymosin beta(4)(Ala), as might be expected from sequence homology. Thymosin beta(4)(Ala) and thymosin beta(10) were found, but thymosin beta(10) is present in about 2.5-fold higher amounts. Because thymosin beta(4)(Ala) possesses about threefold higher affinity to G-actin, compared to thymosin beta(4), beta(10), and beta(9), we suggest that expression of beta-thymosins is triggered by functional requirements and not sequence homology.

Thymosin beta(4) (Tbeta(4)), a highly conserved peptide with immunomodulatory properties, is the major actin-sequestering peptide in mammalian cells. Recent studies have established that Tbeta(4) can accelerate wound healing in full thickness skin wounds and following burn injuries to the cornea. In the eye studies, the accelerated healing due to Tbeta(4) was accompanied by a significant reduction in polymorphonuclear leukocyte (PMN) infiltration and a several-fold decrease in interleukin-1beta (p< or =0.015) and 6-keto-prostaglandin F(1alpha) (6-keto-PGF1alpha, p< or =0.05). Given the recognized role of proinflammatory cytokines in septic shock and of extracellular F- and G-actin in the pathophysiology of multiple organ dysfunction, we have investigated the role of Tbeta(4) in sepsis. We report that an LD(50) dose of LPS (24 mg/kg) in rats resulted in a significant reduction of Tbeta(4) levels in the blood. Furthermore, administration of 100 microg of Tbeta(4) immediately following and at 2 and 4 h after an LD(50) dose of LPS (60 mg/kg) in mice significantly reduced mortality rates (p< or =0.024) and lowered blood levels of a number of inflammatory cytokines, eicosanoids, and other molecules that are highly elevated following endotoxin administration. In studies in human subjects given low doses of endotoxin (4 ng/kg LPS) and in patients with septic shock, we have also observed significant decreases in blood levels of Tbeta(4). The rapid disappearance of Tbeta(4) in the blood following LPS administration or during septic shock suggests that Tbeta(4) may be involved in early events leading to activation of the inflammatory cascade and ultimately the clinical sequelae of sepsis. The results of this study indicate that Tbeta(4) may have utility in the clinic in the treatment of septic shock and in syndromes associated with actin toxicities.

The beta-thymosins constitute a family of highly conserved and extremely water-soluble 5 kDa polypeptides. Thymosin beta4 is the most abundant member; it is expressed in most cell types and is regarded as the main intracellular G-actin sequestering peptide. There is increasing evidence for extracellular functions of thymosin beta4. For example, thymosin beta4 increases the rate of attachment and spreading of endothelial cells on matrix components and stimulates the migration of human umbilical vein endothelial cells. Here we show that thymosin beta4 can be cross-linked to proteins such as fibrin and collagen by tissue transglutaminase. Thymosin beta4 is not cross-linked to many other proteins and its cross-linking to fibrin is competed by another family member, thymosin beta10. After activation of human platelets with thrombin, thymosin beta4 is released and cross-linked to fibrin in a time- and calcium-dependent manner. We suggest that thymosin beta4 cross-linking is mediated by factor XIIIa, a transglutaminase that is coreleased from stimulated platelets. This provides a mechanism to increase the local concentration of thymosin beta4 near sites of clots and tissue damage, where it may contribute to wound healing, angiogenesis and inflammatory responses.

Plasma content of thymosin-alpha(1)and its circadian variations in patients with inflammatory gynecologic diseases differ from those in healthy donors and depend on the type of inflammation and efficacy of treatment. It is concluded that not only the absolute content of thymic hormones, but also their biorhythmic variations are important for immune regulation.

A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise.

Thymosin alpha1 is a cleavage product of prothymosin alpha. Expression of prothymosin alpha increases during cell proliferation. Thymosin alpha1, measurable in plasma, may be an indicator of cell proliferation especially if a cancer is present. In this report we investigated the relation between the clinical behavior of gastric cancer and the plasma thymosin alpha1 level. Plasma thymosin alpha1 was measured in 52 gastric cancer patients using a newly developed radioimmunoassay. Twenty-one tumors and lymph nodes were examined for thymosin alpha1 using immunohistochemistry. The plasma thymosin alpha1 level was higher in gastric cancer patients than in normal volunteers; and it was higher in patients with lymph node involvement than in those with negative nodes. Immunohistochemical study of thymosin alpha1 showed positivity in 52% of gastric cancers and 100% of lymph node metastasis. We concluded that a high level of plasma thymosin alpha1 suggests aggressive behavior of a gastric cancer, such as lymph node involvement.

This paper shows that melatonin regulates both thymosin alpha1 and thymulin production as well as the expression of the prothymosin alpha gene. The results revealed the following facts: (a) The concentrations of thymosin alpha1 in both serum and thymus of rat showed a nyctohemeral profile with peak values late at night and basal values during the day. The concentrations of thymulin in rat serum also showed a 24-h rhythm with an increase in their values at night. This rhythmical character for thymosin alpha1, and thymulin was also found in the human serum. (b) Rats injected with melatonin during the day exhibited a significant increase in the concentrations of both peptides. Moreover, continuous light exposure on the animals at daytime and pinealectomy cause a decrease in thymosin a1 and thymulin concentrations with regards to those found in control rats. (c) Melatonin regulates the expression of the prothymosin alpha gene, analyzed by Northern blot. These results suggest that melatonin may be involved in the regulation of immune functions by increasing the thymic peptides production.

In previous studies, we have shown that castration of Sprague-Dawley rats enhances thymic weight through puberty whilst sex steroids reduce the castration-induced hypertrophy. In the current study, we have confirmed that castration enhances thymic growth compared to age-matched intact controls. In addition, immunoassays were used to measure thymosin alpha1 and thymosin beta4 levels in sera from intact and castrate rats. Castrate animals displayed greater sera levels of thymosins compared to sera from intact animals. To test whether the enhanced thymic weight and increased levels of thymosins observed post-castration were able to influence immune function of castrate animals, concanavalin A was used in the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay to examine lymphocyte and thymocyte responses from both intact and castrate male rats. Responses of cells isolated from castrate rats demonstrated that lymphocytes and thymocytes were stimulated at low levels of concanavalin A (1.56-3.13 microg/ml for lymphocytes and 1.56 microg/ml for thymocytes) compared to the same cell types isolated from intact rats. Concentrations of concanavalin A ranging from 6.25 to 200 microg/ml produced no significant differences in response from intact and castrate animals.

Objective: Thymosin alpha1, an immunomodulatory endogenous peptide, has been shown to be effective in the treatment of chronic hepatitis B and C. In this study, single- and 5-day multiple-dose pharmacokinetics were characterized in nine Caucasian volunteers after subcutaneous administration of 900 microg/m2 thymosin alpha1., Methods: Using a randomized, 3-way crossover design three available drug formulations were compared: Zadaxin (SciClone), Timosina (Sclavo), and Thymosin alpha1 (Tal-HLR; Hoffmann La Roche). AUC, Cmax, t(max), t(1/2), Cl/f, and the volume of distribution, V(Z)/f, were derived by model-independent methods., Results: Endogenous serum concentrations were below the limit of quantification (0.10 microg/l) of the enzyme-linked immunosorbent assay method in most subjects. Thymosin alpha1 was well absorbed with a mean t(max) between 1-2 hours from all galenic formulations. Cmax concentrations of 30 to 80 microg/l and AUC(0-infinity) from 95 to 267 microg x h/l did not differ between single- and multiple-dose administration of all drugs. This apparent lack of accumulation was supported by the short elimination half-life of less than 3 hours. As indicated by a V(Z)/f in the range of 30-40 l, thymosin alpha1 appears to distribute within the extracellular volume. AUC and Cmax were similar for Zadaxin and T alpha1-HLR, but higher after administration of Timosina., Conclusion: Thymosin alpha1 kinetics from this study are comparable to those previously obtained in Japanese volunteers or cancer patients, but may be influenced by the drug formulation used.

The indices of immune status, concentration of alpha 1-thymosin and level of thymus epithelial cell autoantibodies in human blood of persons, who had worked in 30-km zone Chernobyl NPP were studied. The decrease in mean number of leukocytes, lymphocytes, CD5+, CD4+, CD8+ cells, content of serum alpha 1-thymosin and increased level of autoantibodies were detected in workers, whose dose rates irradiation were greater than 25 cSv. So, the quality differences in the changes of immune system, which were induced by low dose chronic irradiation were obtained.

Objective: Prothymosin-alpha, the precursor of thymosin-alpha1, may play a role in cell proliferation, and the plasma level of thymosin-alpha1 may reflect the degree of proliferation of the tumor cells., Methods: Recently, a new sandwich immunoradiometric assay for thymosin-alpha1 was developed using monoclonal and polyclonal antibodies. In this investigation, we used this assay to measure plasma and tissue level of thymosin-alpha1 in 131 lung cancer patients., Results: We found that the mean plasma thymosin-alpha1 levels in lung cancer patients were higher than in normal individuals (P < 0.001). However, half of the patients showed normal levels. Thymosin-alpha1 levels correlated neither with the stage nor pathological subtype of the lung cancer, and did not decrease significantly in the 4 weeks after the resection of the tumor. Thymosin-alpha1 levels of lung cancer patients with another cancer were higher than those without evidence of other cancers (P = 0.03). Survival of patients with normal levels of plasma thymosin-alpha1 was significantly better than that with higher levels (P = 0.04)., Conclusions: The plasma level of thymosin-alpha1 may be used as a marker for the prognosis of lung cancer patients. Further investigations are warranted to determine its role in the lung cancer.

Thymosin fraction 5 (TF5), a thymic preparation, has been shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities. Thymosin alpha1 (T alpha1) was the first biologically active polypeptide to be purified from TF5 and completely characterized. It is an acidic peptide with an isoelectric point of 4.2 and a molecular weight of 3108. T alpha1 is considered a biological response modifier which amplifies T-cell immunity. In the present study, we have studied some pharmacokinetic properties of T alpha1 by measuring its concentrations in serum, urine and ten major organs of female Swiss-Webster mice following administration of 500 microg T alpha1 intraperitoneally. Using a modified enzymatic immunoassay, our data show a significant increase of T alpha1 in serum 2 min after injection and lasting for 2 h (average: 1.55 +/- 0.27 microg/ml). In urine, at four different time points after injection (20 min, 40 min, 2 h, 6 h), increased concentrations of T alpha1 were found between 24.2 and 25.4 microg/ml (average: 25 +/- 0.47 microg/ml). Of the 500 microg T alpha1 administered to mice, 8.97% was recovered at the end of the study, of which 2% corresponded to urine, 1.25% to serum (2 ml of serum per mouse), and 5.72% to organs. Since the urine/day volume and the serum volume of any Swiss Webster mouse is ca 2 ml, additional extrapolation of the above mentioned values could show percentages of recovery close to 40% for urine and 2.5% for serum. In most of the organs, the wet weight concentrations of T alpha1 increased significantly during the first 40 min after injection in comparison to their baseline wet weight concentrations. These organs consisted of the following: thymus (33.1 +/- 3.5 microg/g vs 18 microg/g baseline); lungs (7.7 +/- 1.1 microg/g vs 1.9 microg/g baseline); spleen (15.6 +/- 0.7 microg/g vs 5.6 microg/g); kidneys (6.2 +/- 1.1 microg/g vs 3.9 microg/g); ovaries (9.2 +/- 1.4 microg/g vs 0 microg/g); and peritoneal fat (4 +/- 1 microg/g vs 0 microg/g). No significant increases were observed in the liver (1.7 +/- 0.1 microg/g vs 1.4 microg/g) and heart (0.7 +/- 0.5 microg/g vs 0 microg/g). Increased concentrations of T alpha1 were not detected in the brain and skeletal muscle tissues. These pharmacokinetic studies of T alpha1 in mice indicate that rapid renal excretion of T alpha1 represents a major source of humoral loss following I.P. administration. Recent preliminary studies in humans confirm that the kidney rapidly releases high levels of T alpha1 in urine in a time frame consistent with that observed in mice.

Thymosin beta 4 (T beta 4) is a peptide of 43 amino acids that was first isolated from the thymus gland and subsequently found to be ubiquitous in nature. T beta 4 functions mainly as an actin-sequestering molecule in nonmuscle cells, where its primary role is to maintain the large pool of unpolymerized G-actin in the cell. Studies on the pharmacokinetics of T beta 4 in human and other mammals have not been reported so far. In the present study, we have measured T beta 4 concentrations in serum, urine, and 10 major organs of female Swiss-Webster mice following intraperitoneal administration of 400 micrograms synthetic T beta 4. Using a modified enzymatic immunoassay, our data show a significant increase of T beta 4 in serum starting 2 min after injection and lasting for 40 min (average: 2.34 +/- 0.54 micrograms/ml). High concentrations were found in urine (59.3 +/- 7.54 micrograms/ml) at three different points after injection (20 min, 40 min, and 2 h). Of the 400 micrograms T beta 4 administered to mice 83% was recovered at the end of the study, 44.6% of which corresponded to urine, 1.4% to serum, and 37.5% to the organs. In 50% of the tested organs, the wet weight concentrations of T beta 4 increased significantly from the first 40 min to 2 h after injection in comparison to their baseline wet weight concentrations. These organs were: the brain (72 micrograms/g), heart (80 micrograms/g), liver (15 micrograms/g vs 9 micrograms/g), kidneys (65 micrograms/g vs 28 micrograms/g), and peritoneal fat (47 micrograms/g vs 13 micrograms/g). Wet weight concentrations increased in the thymus (196 micrograms/g vs 147 micrograms/g) and muscle (45 micrograms/g vs 0 micrograms/g) after 6 h of injection. The spleen showed an increase in wet weight concentrations at the 2 min timepoint (267 micrograms/g vs 161 micrograms/g). Ovaries had a biphasic increase at 40 min (72 micrograms/g vs 62 micrograms/g) and 24 h (92 micrograms/g vs 62 micrograms/g) after T beta 4 administration. In lungs, the highest wet weight increase after injection (149 micrograms/g at timepoint 6 h) was not higher than its basal wet weight concentration (153 micrograms/g). These pharmacokinetic studies of T beta 4 in mice have established that high levels of T beta 4 are found in blood following I.P. administration and the kidney rapidly removes the peptide from the circulation. The kinetics of this response should help define the proper scheduling of administration of T beta 4 during clinical trials in disorders, such as the acute respiratory distress syndrome (ARDS), associated with actin toxicity.

We hypothesized that much of the positive effect of porcine somatotropin (pST) on the immune system would be offset by the pST/IGF stimulation of gonadal function and that there would be negative effects on thymic weight and function from increased androgens in males. Male and female hogs (78 boars, 90 gilts) representing three genetic lines (lean, obese, and crossbred meat type) received 0, 2, or 4 mg of recombinant pST/d via implant for 42 d. Blood samples were collected on 0, 7, 14, 28, and 42 d of the trial for changes in pST, IGF-I, IGF-II, thymosin beta 4, dehydroepiandrosterone/dehydroepiandrosterone sulfate (DHEA/DHEASO4), and testosterone concentrations. Thymic, splenic, gonadal, and adrenal weights were collected at slaughter (d 42). Thymic weights increased with dose of pST in both sexes (P < .01), but splenic weights were unaltered. Adrenal weights increased with dose of pST (P < .01), but gonadal weights showed no response to pST in either sex or any line. Overall, concentrations of pST, IGF, and thymosin beta 4 increased with dose of pST. Serum testosterone concentrations in boars declined with dose of pST in lean and obese lines, and DHEA/ DHEASO4 declined in all lines with pST treatment. Testicular testosterone concentrations were not different among lines or doses of pST, indicating no stimulatory effect of increased pST/IGF-I on Leydig cell function. Although pST and its effects through increased circulating IGF are thought to be overall stimulants of growth and protein accretion, the actions on the tissues of the immune system (thymus/spleen) and steroidogenic tissues (adrenal/gonadal) can be selective. Increases in thymic weights and thymosin beta 4 concentrations from pST treatment in boars were partially due to the pST-induced decline in circulatory androgens, and the responses found in this in vivo model do not support pST/IGF effects documented in in vitro systems on Leydig cell function.

A significant decrease in mean number of CD5+, CD8(+)-lymphocytes in persons, who worked in 30-km zone of Chernobyl nuclear power station was revealed. A significant increase in percent of CD5+, CD4(+)-cell percents was observed in workers, who worked for 1,2-2,5 years in zone, but absolute number, were decreased comparing a control and data received in people, who have just arrived to work in 30-km zone. The positive correlation exists between the percent of lymphocytes and years of service in 30-km zone. The lower level of alpha 1-thymosine was revealed in serum of the persons, who worked in zone for 4.5-5 years than data received in people, who worked for 0.5 year. Increase level of serum autoantibodies reacting with thymic epithelial cell was detected in men, who worked in zone for 3-3.5 years. In persons, who worked more 5 years and have just arrived in zone identical data were received.

An Immunoradiometric assay for thymosin alpha 1(1-28) was developed using a monoclonal and a polyclonal antiserum which were raised against synthetic thymosin alpha 1(1-28) and thymosin alpha 1(16-28), respectively. A monoclonal antibody, specific for the (acetylated ser1) 1-5 sequence of thymosin alpha 1, was immobilized on polystyrene beads for the solid phase, and a polyclonal antiserum specific for the 16-28 sequence was employed. This method relies on the formation of an immune complex consisting of a 125I-labelled anti-rabbit IgG goat antibody, polyclonal antiserum, thymosin alpha 1, and the solid phase monoclonal antibody. Radioactivity on the solid phase is directly proportional to the amount of thymosin alpha 1 present in the specimen. The minimal detection limit of this assay system was approximately 1.9 pg/ml. The mean values of thymosin alpha 1 in plasma of healthy subject, ranging in age from 0 to 3 years was approximately five fold higher than that of higher ages. HPLC analysis of plasma of a healthy subjects revealed a single immunoreactive form which eluted with the same retention time as that of synthetic thymosin alpha 1. This assay will be extremely useful for the measurement of thymosin alpha 1 in biologic fluids and tissues.

Thymosin-alpha 1 (thymosin alpha 1) and cortisol levels were evaluated in juvenile squirrel monkeys to investigate the influence of psychological disturbance on thymic and adrenal hormone activity. Hormone levels were assessed in peripheral circulation following removal of monkeys from their social group to establish the time course of thymosin and cortisol alterations. Thymosin alpha 1 was significantly decreased after social separation in association with increased adrenocortical activity, especially during the first day after being housed alone. The temporal pattern suggested that both hormone systems are involved in the acute inhibition of functional immunity observed following this type of psychological disturbance. A second study verified that the decrement in thymosin alpha 1 levels was replicable and also sensitive to psychosocial factors that influence the level of induced disturbance. In addition, changes in thymosin secretion could be attenuated partially by pharmacological inhibition of the cortisol response and opiate hormone action. Nevertheless, the decrease in thymosin alpha 1 secretion did not appear to be a secondary consequence of adrenocortical secretion, and instead probably emanates from a general shift in neuroendocrine activity.

Elucidation of cause and effect relationships between gonadal and thymic function can be particularly challenging due to the complexity of interactions among various hormones on thymic function, many of which are largely unknown. To monitor gonadal-thymic interaction with the endocrine changes associated with maturity, bulls, steers, heifers, and ovariectomized heifers were slaughtered at 4, 6, 10, 12, or 14 months of age (n = 5-7/sex/age). Steers and ovariectomized heifers were castrated before 1 week of age. Thymic weights were increased in castrated animals as early as 4 months of age (p < 0.01), and differences were more pronounced in males. Thymic secretory peptide, thymosin beta4, increased up to six months of age in all groups (p < 0.05) and then decreased with age, similar to the pattern of growth hormone. Prepuberal testosterone concentrations increased to 10-12 months of age and then decreased in both bulls and heifers (p < 0.05), coordinate with decreases of thymosin beta4 at 10 months of age. Growth hormone concentrations increased up to 6 months of age in all groups (p < 0.01) and then declined. Concentrations of prolactin peaked at 10 months of age (p < 0.01) but were not different between groups. Relationships of luteinizing hormone (LH) were highly related to thymosin beta4 changes in heifers during maturation. Changes of thymosin beta4 and LH were coordinate in ovary-intact heifers and inversely related in ovariectomized heifers. Gonadal effects on thymic weight were evident as early as 4 months of age, and no abrupt declines in thymic weight were detected at puberty.

Irradiation of the epithelial cells from human thymus (cell line HTSC) by arsenid-gallium laser (0.89 nm, 1500 Hz) induces an enhancement of thymic hormone production. The concentration of alpha-1-thymosin was substantially increased on the days 1 and 5 in culture after irradiation. The increase of thymulin level in cultural supernatants was less pronounced and displayed after irradiation during 3-4 minutes. Local laser irradiation of thymus region of rats (5 minutes, 10 times) induce the increase of serum concentration of alpha-1-thymosin and the decrease of serum level of thymulin 2 days after irradiation.

Experiment 1 involved genetically lean and obese gilts and barrows injected for 42 d with 0, 2, or 4 mg/d of porcine somatotropin (pST; n = 96). Blood was collected at 0, 6, and 24 h after the initial pST injection and thereafter every 2 wk. Experiment 2 involved lean and obese gilts and barrows (n = 48) that were administered implants that released 0, 2, or 4 mg/d of pST for 42 d. Blood was collected at d 0, 7, 14, 28, and 42 of the study. Thymic weights and a final blood sample were collected at the end of the 42-d trial. Thymic weights increased (P < .01) with dose of pST in injected and implanted animals. Thymosin beta 4 increased (P < .05) in a pST dose-dependent manner in injected and implanted animals, but concentrations were increased more in implanted animals. Concentrations of thymosin beta 4 decreased or reached a plateau in gilts but continued to increase in barrows to the end of the trial. Thymosin alpha 1 concentrations were increased (P < .01) in barrows compared with gilts, but the concentrations were not related to dose of pST. Concentrations of pST in injected animals increased (P < .01) within 6 h after injection and had returned to basal concentrations by 24 h after injection. Concentrations of pST in implanted animals were dose-dependent (P < .05) and remained increased throughout the trial. Concentrations of IGF-I and -II were pST-dependent (P < .01) and increased throughout the trial period. Outside of the pST dose relationships, temporal changes in thymosin beta 4 were not highly related to changes in pST, IGF-I, or IGF-II. The pST dose-related increases in thymic weights and thymosin beta 4 concentrations are consistent with pST stimulation of the immune function in conjunction with overall increases of growth and efficiency of live weight gain in swine.