Neutrophil activation in septic acute kidney injury: A post hoc analysis of the FINNAKI study.

BACKGROUND: Inflammation, reflected by high plasma interleukin-6 concentration, is associated with acute kidney injury (AKI) in septic patients. Neutrophil activation has pathophysiological significance in experimental septic AKI. We hypothesized that neutrophil activation is associated with AKI in critically ill sepsis patients. METHODS: We measured plasma (n = 182) and urine (n = 118) activin A (a rapidly released cytosolic neutrophil protein), interleukin-8 (a chemotactic factor for neutrophils), myeloperoxidase (a neutrophil biomarker released in tissues), and interleukin-6 on intensive care unit admission (plasma and urine) and 24 hours later (plasma) in sepsis patients manifesting their first organ dysfunction between 24 hours preceding admission and the second calendar day in intensive care unit. AKI was defined by the Kidney Disease: Improving Global Outcomes criteria. RESULTS: Plasma admission interleukin-8 (240 [60-971] vs 50 [19-164] pg/mL, P < .001) and activin A (845 [554-1895] vs 469 [285-862] pg/mL, P < .001) were but myeloperoxidase (169 [111-300] vs 144 [88-215] ng/mL, P = .059) was not higher among patients with AKI compared with those without. Urine admission interleukin-8 (50.4 [19.8-145.3] vs 9.5 [2.7-28.7] ng/mL, P < .001) and myeloperoxidase (7.7 [1.5-12.6] vs 1.9 [0.4-6.9] ng/mL, P < .001) were but activin A (9.7 [1.4-42.6] vs 4.0 [0.0-33.0] ng/mL, P = .064) was not higher in AKI than non-AKI patients. Urine myeloperoxidase correlated with urine interleukin-8 (R = .627, P < .001) but not with plasma myeloperoxidase (R = .131, P = .158). CONCLUSION: Interleukin-8 in plasma and urine was associated with septic AKI. Elevated plasma activin A indicates intravascular neutrophil activation in septic AKI. Concomitant plasma and urine myeloperoxidase measurements suggest neutrophil accumulation into injured kidneys.

Seminal plasma transforming growth factor-ß, activin A and follistatin fluctuate within men over time.

STUDY QUESTION: Do seminal plasma transforming growth factor-ß (TGFB) cytokines vary within individuals over time, and does this relate to sperm parameters, age or prior abstinence? SUMMARY ANSWER: Activin A and follistatin, and to a lesser extent TGFB1, TGFB2 and TGFB3, vary within individuals over time, in association with duration of abstinence. WHAT IS ALREADY KNOWN: Seminal plasma TGFB cytokines can influence sperm function and reproductive success through interactions with the female reproductive tract after coitus. Over time, individual sperm parameters fluctuate considerably. Whether seminal fluid TGFB cytokines vary similarly, and the determinants of any variance, is unknown. STUDY DESIGN, SIZE, DURATION: Between two and seven semen samples were collected from each of 14 fertile donors at 6-10 week intervals over the course of 12 months, then seminal plasma cytokines and sperm parameters were measured. PARTICIPANTS/MATERIALS, SETTING AND METHOD: The concentrations and total amounts per ejaculate of TGFB1, TGFB2, TGFB3, activin A and follistatin were determined using commercial assays. Sperm parameters were assessed according to WHO IV standards. Mixed model analysis was utilised to determine the relative contribution of between- and within-individual factors to the variance. Relationships between cytokines and sperm parameters, as well as effect of age and duration of abstinence, were investigated by correlation analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Within-individual variability contributed to the total variance for all cytokines and sperm parameters, and was a stronger determinant than between-individual variability for activin A and follistatin as well as for total sperm concentration and sperm motility. Positive correlations between each of the three TGFB isoforms, and activin and follistatin, suggest co-regulation of synthesis. Duration of abstinence influenced total content of TGFB1, TGFB2, activin A and follistatin. TGFB1 correlated inversely with age. LIMITATIONS, REASONS FOR CAUTION: A limited number of donors from a single clinic were investigated. Clinical information on BMI, nutrition, smoking and other lifestyle factors was unavailable. Further studies are required to determine whether the findings can be generalised to larger populations and different ethnicities. WIDER IMPLICATIONS OF THE FINDINGS: These data reveal substantial variation over time in seminal fluid cytokines and indicate that repeated analyses are required to gain precise representative data on an individual's status. Within-individual variation in seminal fluid components should be taken into account when investigating seminal fluid cytokines. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the National Health and Medical Research Council of Australia, ID453556 and APP1041332. The authors have no competing interests to disclose.

Expression of Cripto-1 gene protein and Activin-A in human lung adenocarcinoma tissue.

To research the expression in human lung adenocarcinoma tissue of Cripto-1 (teratocarcinoma derived growth factor-1) gene protein and Activin-A gene protein, and explore the relationship and clinical significance between the two gene protein and clinical pathological characteristic of lung adenocarcinoma. This study had applied the immunohistochemical method to detect the 188 cases of lung adenocarcinoma and expression of Cripto-1 protein and Activin-A protein in 100 cases of normal lung tissue. Then, analysis the relationship between these two-gene protein and clinical lung adenocarcinoma histopathological features, and inherent correlation between these two genes. The positive expression rate of Cripto-1 protein in lung adenocarcinoma tissue was significantly higher in normal lung tissue, while, the positive expression rate of Activin-A protein in lung adenocarcinoma tissue was significantly lower than in normal lung tissue. The high expression of Cripto-1 and low expression of Activin-A was closely related (each P<0.05) to the TNM staging of lung adenocarcinoma, lymph node metastasis and the main pathological tissue staging of lung adenocarcinoma. And the correlation analysis showed that it was negative correlation for the expression of Activin-A protein and Cripto-1 protein in lung adenocarcinoma. The over expression of Cripto-1 and the expression lack of Activin-A were correlated with the occurrence, development, metastasis and malignant degree of lung adenocarcinoma.

Activin-A overexpression in the murine lung causes pathology that simulates acute respiratory distress syndrome.

RATIONALE: Activin-A is up-regulated in various respiratory disorders. However, its precise role in pulmonary pathophysiology has not been adequately substantiated in vivo. OBJECTIVES: To investigate in vivo the consequences of dysregulated Activin-A expression in the lung and identify key Activin-A-induced processes that contribute to respiratory pathology. METHODS: Activin-A was ectopically expressed in murine lung, and functional, structural, and molecular alterations were extensively analyzed. The validity of Activin-A as a therapeutic target was demonstrated in animals overexpressing Activin-A or treated with intratracheal instillation of LPS. Relevancy to human pathology was substantiated by demonstrating high Activin-A levels in bronchoalveolar lavage (BAL) samples from patients with acute respiratory distress syndrome (ARDS). MEASUREMENTS AND MAIN RESULTS: Overexpression of Activin-A in mouse airways caused pulmonary pathology reminiscent of acute lung injury (ALI)/ARDS. Activin-A triggered a lasting inflammatory response characterized by acute alveolar cell death and hyaline membrane formation, sustained up-regulation of high-mobility group box 1, development of systemic hypercoagulant state, reduction of surfactant proteins SpC, SpB, and SpA, decline of lung compliance, transient fibrosis, and eventually emphysema. Therapeutic neutralization of Activin-A attenuated the ALI/ARDS-like pathology induced either by ectopic expression of Activin-A or by intratracheal instillation of LPS. In line with the similarity of the Activin-A-induced phenotype to human ARDS, selective up-regulation of Activin-A was found in BAL of patients with ARDS. CONCLUSIONS: Our studies demonstrate for the first time in vivo the pathogenic consequences of deregulated Activin-A expression in the lung, document novel aspects of Activin-A biology that provide mechanistic explanation for the observed phenotype, link Activin-A to ALI/ARDS pathophysiology, and provide the rationale for therapeutic targeting of Activin-A in these disorders.

Preeclampsia and Its Impact on Human Milk Activin A Concentration.

BACKGROUND: It is known that preeclampsia affects lactogenesis. However, data on the effects of this pathology on human milk neurobiomarker composition are not available. The aim of this study is to investigate the effects of this gestational pathology on activin A levels, a neurobiomarker known to play an important role in the development and protection of the central nervous system. METHODS: The women recruited were divided in two different study groups: preeclamptic or normotensive women. All the human milk samples were collected using the same procedure. Activin A was quantified using an Enzyme-linked immunosorbent assay (ELISA) test. To investigate the effect of preeclampsia on the activin A concentration in the three lactation phases, a mixed linear model with a unistructural covariance structure, with the mother as the random effect, and fixed effects were performed. RESULTS: Activin A was detected in all samples. There were no significant differences between preeclamptic and normotensive women. The only significant effect is related to the lactation phase: the difference between colostrum and mature milk (p < 0.01) was significant. In conclusion, these results allow us to affirm that breast milk's beneficial properties are maintained even if preeclampsia occurs.

Growth factor expression pattern of homologous feeder layer for culturing buffalo embryonic stem cell-like cells.

The present study examined the expression profile of buffalo fetal fibroblasts (BFF) used as a feeder layer for embryonic stem (ES) cell-like cells. The expression of important growth factors was detected in cells at different passages. Mitomycin-C inactivation increased relative expression levels of ACTIVIN-A, TGF-ß1, BMP-4 and GREMLIN but not of fibroblast growth factor-2 (FGF-2). The expression level of ACTIVIN-A, transforming growth factor-ß1 (TGF-ß1), bone morphogenetic protein-4 (BMP-4) and FGF-2 was similar in buffalo fetal fibroblast (BFF) cultured in stem cell medium (SCM), SCM+1000IU mL(-1) leukemia inhibitory factor (LIF), SCM+5 ngmL(-1) FGF-2 or SCM+LIF+FGF-2 for 24 h whereas GREMLIN expression was higher in FGF-2-supplemented groups. In spent medium, the concentration of ACTIVIN-A was higher in FGF-2-supplemented groups whereas that of TGF-ß1 was similar in SCM and LIF+FGF-2, which was higher than when either LIF or FGF-2 was used alone. Following culture of ES cell-like cells on a feeder layer for 24 h, the TGF-ß1 concentration was higher with LIF+FGF-2 than with LIF or FGF-2 alone which, in turn, was higher than that in SCM. In the LIF+FGF-2 group, the concentration of TGF-ß1 was lower and that of ACTIVIN-A was higher in spent medium at 24 h than at 48 h of culture. These results suggest that BFF produce signalling molecules that may help in self-renewal of buffalo ES cell-like cells.

Seasonal changes in immunoreactivity of activin signaling component proteins in wild ground squirrel testes.

The seasonal spermatogenesis and localization of inhibin/activin subunits (alpha, betaA, betaB) in the testes of wild ground squirrel has been previously described; however, the expression pattern of activin receptors and cytoplasmic signaling SMADs has not been detected in any seasonal breeders. The objective of this study was to investigate the abundance and cellular localization of activin signaling components in testes of the wild ground squirrel during the breeding and nonbreeding seasons. The immunolocalizations of ActRIIB (activin type II receptor B) and activin-related SMADs (phospho-SMAD2/3, SMAD4 and SMAD7) were observed by immunohistochemistry. Total proteins were extracted from testicular tissues in the breeding and nonbreeding seasons and were used for Western blotting analysis for ActRIIB and SMADs. Immunoreactivities of activin signaling components were greater in the testes of the breeding season, and then decreased to a relatively low level in the nonbreeding season. ActRIIB and related SMADs were widely spread in the active testes, while spermatogonia were the predominant cellular sites of activin signal transduction during arrested spermatogenesis. The dynamic regulation of activin type II receptor and SMADs indicated that the activin signal pathway played an important paracrine role in seasonal spermatogenesis of the wild ground squirrel. Furthermore, the distinct localizations and immunoreactivity of ActRIIB and SMADs might suggest different functions of activin in seasonal spermatogenesis.

Detection of activin receptor type IIA and IIB-Fc fusion proteins by automated capillary immunoassay.

Activin receptor type IIA and type IIB fusion protein have been designed to sequester circulating molecules of the transforming growth factor-ß (TGF-ß) superfamily and inactivate their actions. Members of this superfamily have been reported as essential regulators of erythropoiesis by triggering the formation of activated ternary complexes containing different combinations of type I and type II receptors, which can limit RBC production by accelerating erythroid differentiation and inhibiting erythroid progenitor expansion. The recent approval of Luspatercept for the treatment of anemia associated to transfusion-dependent MDS and Beta-thalassemia in afflicted patients means that it can now pose a real threat of being abused in sport for its ability to stimulate erythropoiesis. Several methods for the detection of these molecules in blood have been proposed for the purpose of sport antidoping control. Here we propose the detection of the ActRIIA-Fc and ActRIIB-Fc fusion proteins by automated capillary Western immunoassay (Simple Western). The use of these immunoassays for the detection of protein targets has become widespread in the recent years. The work presented here demonstrates that this methodology enables a versatile, rapid, and sensitive detection of activin ligand traps in blood samples: plasma, serum, or dried blood spots (DBS). Preliminary results indicate that detection in urine samples is also possible. The option to use different antibodies allows the possibility to use this method as an initial testing procedure as well as a confirmation procedure. Finally, results coming from an administration study confirm that the method is suitable for routine analysis.

Anti-inflammatory therapy in an ovine model of fetal hypoxia induced by single umbilical artery ligation.

Perinatal morbidity and mortality are significantly higher in pregnancies complicated by chronic hypoxia and intrauterine growth restriction (IUGR). Clinically, placental insufficiency and IUGR are strongly associated with a fetoplacental inflammatory response. To explore this further, hypoxia was induced in one fetus in twin-bearing pregnant sheep (n=9) by performing single umbilical artery ligation (SUAL) at 110 days gestation. Five ewes were administered the anti-inflammatory drug sulfasalazine (SSZ) daily, beginning 24h before surgery. Fetal blood gases and inflammatory markers were examined. In both SSZ- and placebo-treated ewes, SUAL fetuses were hypoxic and growth-restricted at 1 week (P<0.05). A fetoplacental inflammatory response was observed in SUAL pregnancies, with elevated pro-inflammatory cytokines, activin A and prostaglandin E(2). SSZ did not mitigate this inflammatory response. It is concluded that SUAL induces fetal hypoxia and a fetoplacental inflammatory response and that SSZ does not improve oxygenation or reduce inflammation. Further studies to explore whether alternative anti-inflammatory treatments may improve IUGR outcomes are warranted.

Serum activin A levels are lower in tubal than intrauterine spontaneously conceived pregnancies.

OBJECTIVE: To measure serum activin A levels together with progesterone and hCG, in women with overt clinical signs and symptoms of ectopic pregnancy (EP) and, in gestational age-matched intrauterine pregnancy (IUP). DESIGN: Retrospective case-control study. SETTING: Department of Pediatrics, Obstetrics and Reproductive Medicine, Section of Obstetrics and Gynecology, University of Siena, Siena, Italy. POPULATION: The study group was composed by 30 women with an EP; the control group was composed by 30 women with a sonographic evidence of a single spontaneous IUP. METHODS: Clinical examination; transvaginal ultrasound scan; hCG, progesterone and activin-A measurements; laparoscopy; uterine curettage; histological examination. MAIN OUTCOME MEASURE: Pregnancy outcome; sensitivity and specificity of hCG, progesterone, and activin A for EP. RESULTS: Serum hCG levels did not differ significantly between tubal EP and IUP, while P concentrations were significantly (P < 0.001) lower in tubal EP than IUP. Serum levels of activin A were significantly (P < 0.0001) lower in tubal EP than in IUP and, at the cutoff 0.43 ng/mL achieved a sensitivity of 96.7% and a specificity of 100% for EP. CONCLUSION: Activin A secretion in EP is reduced and measurement of its serum levels may have the potential clinical advantage to signal the presence of EP.

Potential biomarkers of temporomandibular joint disorders.

PURPOSE: The purpose of this study was to identify protein markers present in subjects with temporomandibular joint disorders (TMDs) and clicking compared with the levels in controls. MATERIALS AND METHODS: This was a pilot case-control study, and we report the preliminary results. Samples of joint aspirate collected from patients with TMDs and controls who had undergone surgery for a problem other than TMDs were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and biotin-labeled-based protein arrays. The data obtained from these techniques were used to identify the proteins of interest, which were then quantitated using enzyme-linked immunosorbent assay (ELISA). The patient samples studied included joint aspirate collected clinically from the controls and patients and included samples from both the right and the left sides of each patient with a TMD. RESULTS: The 8 TMJ aspirate samples from 6 subjects included 5 aspirate samples from 4 patients and 3 from 2 controls. The greatest standardized protein concentration of endocrine gland-derived vascular endothelial growth factor/prokineticin-1 (EG-VEGF/PK1) and D6 was found in both joints of the controls compared with the levels from the joints of the patients. With 1 exception, the standardized protein concentration was significantly lower in the patients than in the controls. The lower levels of EG-VEGF/PK1 and D6 in the patients compared with the controls suggest that these cytokines might be possible biomarkers for TMDs. CONCLUSION: In the present pilot study, greater levels of EG-VEGF/PK1 and D6 were found in the controls than in the patients with TMDs. Proteomic analysis of the proteins present in the diseased joints compared with those in the controls might help to identify proteins present when pain or degeneration of the joint occurs. The proteomic information might be useful in the development of future therapies.

Testicular immune cell populations and macrophage polarisation in adult male mice and the influence of altered activin A levels.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/-) or elevated activin A (Inha+/-) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII- correspondingly higher in Inha+/- testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.

Activin A triggers angiogenesis via regulation of VEGFA and its overexpression is associated with poor prognosis of oral squamous cell carcinoma.

Poor prognosis associated with the dysregulated expression of activin A in a number of malignancies has been related to with numerous aspects of tumorigenesis, including angiogenesis. The present study investigated the prognostic significance of activin A immunoexpression in blood vessels and cancer cells in a number of oral squamous cell carcinoma (OSCC) cases and applied in vitro strategies to determine the impact of activin A on angiogenesis. In a cohort of 95 patients with OSCC, immunoexpression of activin A in both blood vessels and tumor cells was quantified and the association with clinicopathological parameters and survival was analyzed. Effects of activin A on the tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs) were evaluated in gain­of­function (treatment with recombinant activin A) or loss­of­function [treatment with activin A­antagonist follistatin or by stable transfection with short hairpin RNA (shRNA) targeting activin A] conditions. Conditioned medium from an OSCC cell line with shRNA­mediated depletion of activin A was also tested. The profile of pro­ and anti­angiogenic factors regulated by activin A was assessed with a human angiogenesis quantitative PCR (qPCR) array. Vascular endothelial growth factor A (VEGFA) and its major isoforms were evaluated by reverse transcription­qPCR and ELISA. Activin A expression in blood vessels demonstrated an independent prognostic value in the multivariate analysis with a hazard ratio of 2.47 [95% confidence interval (CI), 1.30­4.71; P=0.006) for disease­specific survival and 2.09 (95% CI, 1.07­4.08l: P=0.03) for disease­free survival. Activin A significantly increased tubular formation of HUVECs concomitantly with an increase in proliferation. This effect was validated by reduced proliferation and tubular formation of HUVECs following inhibition of activin A by follistatin or shRNA, as well as by treatment of HUVECs with conditioned medium from activin A­depleted OSCC cells. Activin A­knockdown increased the migration of HUVECs. In addition, activin A stimulated the phosphorylation of SMAD2/3 and the expression and production of total VEGFA, significantly enhancing the expression of its pro­angiogenic isoform 121. The present findings suggest that activin A is a predictor of the prognosis of patients with OSCC, and provide evidence that activin A, in an autocrine and paracrine manner, may contribute to OSCC angiogenesis through differential expression of the isoform 121 of VEGFA.

A new 'total' activin B enzyme-linked immunosorbent assay (ELISA): development and validation for human samples.

BACKGROUND AND OBJECTIVE: There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a 'total' activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs. DESIGN: The new ELISA was validated and then used to measure activin B levels in the circulation of healthy participants, IVF patients, pregnant women and in ovarian follicular fluid and seminal plasma. PATIENTS AND MEASUREMENTS: Healthy adult subjects (n = 143), subjects from an IVF clinic (n = 27) and pregnancy groups (n = 29) were sampled. RESULTS: The sensitivity of the assay was 0.019 ng/ml. Validation of the activin B ELISA showed good recovery (90.7 +/- 9.8%) and linearity in biological fluid and cell culture media and low cross-reactivity with related analytes (inhibin B = 0.077% and activin A = 0.0034%). There was a negative correlation between activin B concentration (r = -0.281, P < 0.011) and females with increasing age. Patients attending IVF clinics had significantly lower levels of activin B compared with gender-matched control subjects. Ovarian follicular fluid and seminal plasma had 50-80 fold higher levels of activin B (mean = 5.35 and 3.66 ng/ml respectively) than sera (mean = 0.071 ng/ml). CONCLUSIONS: This fully validated ELISA for activin B offers a tremendous utility for measuring this protein in a variety of normal physiological processes and in various clinical pathologies.

Placental production and maternal serum and urine levels of inhibin A and activin A are modified by antihypertensive therapy in hypertensive disorders of pregnancy.

OBJECTIVE: Levels of inhibin A and activin A are raised in pre-eclampsia (PE) but it is not known if antihypertensive therapy can affect their levels. Our aim was to investigate the effect of the antihypertensive drug alpha-methyldopa on serum, urine and placental concentrations of inhibin A and activin A in women presenting with hypertensive disorders of pregnancy. DESIGN: This was a cross-sectional study. PATIENTS: We recruited 65 women presenting with PE, 39 with gestational hypertension (GH) and 104 normotensive controls matched for maternal age, gestational age and parity. MEASUREMENTS: Using specific validated ELISAs, serum and urine levels of inhibin A and activin A, and uterine artery Doppler indices, were measured before and 24-48 h after initiating alpha-methyldopa therapy in women with PE, with GH and controls. Protein extracts were obtained from samples of placental tissue from another group of women with PE, GH and controls for the same analysis. RESULTS: In PE, but not GH, alpha-methyldopa therapy was associated with significantly (P < 0.05) lower levels of both serum and urine inhibin A and activin A. Similarly, in PE but not GH, alpha-methyldopa therapy was associated with lower placental levels of both markers (P < 0.05). There was no significant difference in pulsatility index following treatment in either PE or GH. CONCLUSIONS: Our data indicate that antihypertensive therapy with alpha-methyldopa may have an effect on the synthesis and/or release of placental proteins in pregnancies complicated by PE and that this effect may be independent of its known antihypertensive action.

Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas.

Activin is a pleiotropic growth factor belonging to the transforming growth factor-beta (TGFB) superfamily of signaling molecules. Regulated activin signaling is known to influence several steps in rodent male gamete differentiation. TGFB ligand isoforms, TGFB1-B3, also influence germ cell survival in the rodent testis at the onset of spermatogenesis and around the time of puberty. Given the importance of regulated activin and TGFB signaling in testis development and function, we sought to investigate the cellular production sites of activin/TGFB-signaling modulators in normal and dysfunctional adult human testes samples. Signaling transducers phosphorylated SMAD2/3, and signaling modulators SMAD6, MAN-1, inhibin alpha (INHA), and beta-glycan were detected in Bouins fixed, paraffin-embedded adult human testis sections using immunohistochemistry. Additional samples examined were from testicular cancer patients and from normal men subjected to gonadotropin suppression with androgen-based contraceptives. Our findings identify distinct differences between normal and gonadotropin-deprived human testis in the expression and cellular localization of activin/TGFB-signaling modulators. The presence of a nuclear phosphorylated SMAD2/3 signal in all analyzed seminoma specimens indicated active activin/TGFB signaling. Moreover, a subset of seminoma specimens exhibited selective enhanced expression of beta-glycan (4 out of 28 seminoma tumors), INHA (6 out of 28), and MAN-1 (6 out of 28), highlighting potential functional differences between individual tumors in their capacity to regulate activin/TGFB signaling. Within the heterogenous nonseminomas, expression of signaling modulators was variable and reflected the degree of somatic differentiation. Thus, synthesis of activin and TGFB-signaling modulators may be affected by spermatogenic disruption and altered hormone levels in the testis.

Immunohistochemical localization of inhibin/activin subunits in adult Asian elephant (Elephas maximus) testes.

Immunolocalization of inhibin-α and inhibin/activin ßA and ßB subunits in the testes of Asian elephant was determined. Testicular sections were immunostained with polyclonal antisera against inhibin subunit-α and inhibin/activin ßA and ßB using the avidin-biotin-peroxidase complex method. Positive immunostaining against inhibin-α subunit was strongly present in Sertoli cells, and positive immunostaining for the inhibin/activin ßA and ßB subunits was observed in both Sertoli and Leydig cells. These results indicated that while Sertoli cells are the predominant source of inhibin and activin secretions in the testes of adult male Asian elephant, Leydig cells are a source of activin but not inhibin.

Role of Activin A in the Pathogenesis of Endothelial Cell Dysfunction in Preeclampsia.

This chapter describes the methodologies which may be used in evaluating in vitro endothelial cell dysfunction in preeclampsia.

Lutein levels in arterial cord blood correlate with neuroprotein activin A in healthy preterm and term newborns: A trophic role for lutein?

BACKGROUND: Lutein (LT) is a naturally occurring xanthophyll carotenoid most predominant in the central nervous system (CNS), but its neurotrophic role is still debated. We therefore investigated whether cord blood concentrations correlated with a well-established neurobiomarker, namely activin A. METHODS: We conducted a prospective study on the distribution of LT and activin A in arterial cord blood of healthy preterm (n=50) and term (n=82) newborns according to weeks of gestational age (wGA) and gender. RESULTS: LT and activin A showed a pattern of concentration characterized by higher levels (P<0.01, for all) at 33-36 wGA followed by a progressive decrease (P<0.01, for all) from 37 onwards with a dip at term. Both LT and activin A were gender-dependent with significantly (P<0.01, for all) higher levels in all recruited females and after sub-grouping for preterm and term births. LT (R=0.33; P<0.001) correlated with wGA at sampling. There were significant positive correlations between lutein and activin A in male (R=0.93; P<0.001) and female (R=0.89; P<0.001) groups. CONCLUSIONS: The present data showing a correlation between LT and activin A support the notion of a neurotrophic role gender-dependent for LT and open the way to further investigations correlating LT with well-established biochemical markers of CNS development/damage.

Combined detection of the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc) in serum by means of immunoaffinity purification, tryptic digestion, and LC-MS/MS.

Therapeutic proteins are a continuously growing class of pharmaceuticals and comprise several drug candidates with potential performance-enhancing properties. In particular, activin receptor competitors, such as the ActRII-Fc fusion proteins Sotatercept (ActRIIA-Fc) and Luspatercept (modified ActRIIB-Fc), have the potential for being misused as doping agents in sports as they were found to inhibit negative regulators of late-stage erythropoiesis. Within this study, ammonium sulfate precipitation, immunoaffinity purification, tryptic digestion, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to develop an assay for the combined detection of Sotatercept and Luspatercept in doping control serum samples. The assay was optimized, comprehensively characterized, and found to be fit-for-purpose for application to sports drug testing. It complements existing tests for ActRII-Fc fusion proteins and expands the range of available detection methods for novel protein therapeutics.