Effect of yeast cell wall supplementation on intestinal integrity, digestive enzyme activity and immune traits of broilers.

1. The protective layer formed by intestinal epithelial cells acts as a barrier preventing the adhesion of pathogenic bacteria, aids digestion and passage of nutrients and reduces damage caused from toxins on the gastrointestinal tract. This study was conducted to investigate the effects of a yeast cell wall-based product (YCW), on broiler intestinal integrity, digestive enzyme capacity and immune function.2. A 35-d trial involving 246, one-d-of-hatch male broiler chickens was carried out at a trial facility at Agri-Food Biosciences Institute (AFBI, Belfast, UK). Birds were randomly allocated into 6 pens at day of hatch (41 birds/pen; 123 birds/group). Pens were divided into two groups: (1) basal diet and (2) basal diet that incorporated YCW at the manufacturers' recommended inclusion levels (Alltech Inc., Lexington, Kentucky, USA).3. In this study, YCW supplementation affected broiler intestinal morphology resulting in greater crypt depth, villus height and surface area, goblet cell density and mucus layer thickness and lower muscularis mucosae thickness. The digestive enzymes, maltase, sucrase and alkaline phosphatase, were significantly higher in the YCW supplemented group compared to the control. The expression levels of pro-inflammatory cytokines, IL-1ß, IL-12 and IL-18, were significantly lower as was necroptotic cell death in YCW supplemented birds.4. In conclusion, under the conditions of this study, YCW supplementation positively affected intestinal health parameters in broilers following 35-d supplementation.

TNF-α and its soluble receptors mediate the relationship between prior severe mood episodes and cognitive dysfunction in euthymic bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is one of the most disabling mental health conditions in the world. Symptoms of cognitive impairment in BD contribute directly to occupational and social deficiencies and are very difficult to treat. Converging evidence suggests that BD patients have increased peripheral markers of inflammation. The hypothesis of neuroprogression in BD postulates that cognitive deficits develop over the course of the illness and are influenced by prior severe mood episodes, leading to wear-and-tear on the brain- however, there exists a paucity of data statistically testing a mediating role of immune molecules in cognitive dysfunction in BD. METHODS: This is a cross-sectional study. We measured serum levels of tumor necrosis factor alpha (TNF-α), and soluble (s) TNF receptors one and two (sTNF-R1 and sTNF-R2) in 219 euthymic BD patients and 52 Healthy Controls (HCs). Structural equation modeling (SEM) was used for the primary purpose of assessing whether TNF markers (measured by the multiple indicators TNF-α, sTNF-R1 and sTNF-R2) mediate the effect or number of prior severe mood episodes (number of prior psychiatric hospitalizations) on cognitive performance. RESULTS: BD and HC groups did not differ on circulating levels of TNF molecules in the present study. However, we found higher sTNF-R1 concentration in 'late-stage' BD illness (>1 prior psychiatric hospitalization) compared to those in early stage illness. In the subsequent SEM, we found that the model fits the data acceptably (Chi-square = 49.2, p = 0.3), and had a 'close fit' (RMSEA = 0.02, PCLOSE = 0.9). Holding covariates constant (age, sex, premorbid IQ, education, and race), we found that the standardized indirect effect was significant, p = 0.015, 90%CI [-0.07, -0.01], indicating that the estimated model was consistent with peripheral TNF markers partially mediating a causal effect of severe mood episodes on executive function. CONCLUSIONS: Our results indicate that circulating levels of TNF molecules partially mediate the relationship between prior severe mood episodes and executive function in BD. These results may implicate TNF variables in the neuroprogressive course of BD and could point to novel interventions for cognition.

Phylogenetic and functional alterations in bacterial community compositions in broiler ceca as a result of mannan oligosaccharide supplementation.

This study focused on identifying reproducible effects of dietary supplementation with a mannan oligosaccharide (MOS) on the broiler cecal bacterial community structure and function in a commercial production setting. Two separate trials, each with a control and a supplemented group, were carried out in the same commercial location and run concurrently. Approximately 10,000 birds from the same commercial hatchery were mirror imaged into each of four commercial broiler sheds and fed either a control or supplemented diet. Cecal contents were obtained on days 7, 21, and 35 posthatch from 12 randomly caught broilers from each group. Bacterial pyrosequencing was performed on all samples, with approximately 250,000 sequences obtained per treatment per time point. The predominant phyla identified at all three time points in both trials were Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Tenericutes, representing >99% of all sequences. MOS supplementation altered the bacterial community composition from 7 days supplementation through 35 days supplementation. Bacteroidetes appeared to be replacing Firmicutes as a result of supplementation, with the most noticeable effects after 35 days. The effects of supplementation were reproducible across both trials. PICRUSt was used to identify differences between the functional potentials of the bacterial communities as a result of MOS supplementation. Using level 3 KEGG ortholog function predictions, differences between control and supplemented groups were observed, with very strong segregation noted on day 35 posthatch in both trials. This indicated that alterations of bacterial communities as a result of MOS are likely to alter the functional capability of the cecum.

Pharmacogenetics of pemetrexed combination therapy in lung cancer: pathway analysis reveals novel toxicity associations.

Identification of polymorphisms that influence pemetrexed tolerability could lead to individualised treatment regimens and improve quality of life. Twenty-eight polymorphisms within eleven candidate genes were genotyped using the Illumina Human Exome v1.1 BeadChip and tested for their association with the clinical outcomes of non-small cell lung cancer and mesothelioma patients receiving pemetrexed/platinum doublet chemotherapy (n=136). GGH rs11545078 was associated with a reduced incidence of grade ⩾3 toxicity within the first four cycles of therapy (odds ratio (OR) 0.25, P=0.018), as well as reduced grade ⩾3 haematological toxicity (OR 0.13, P=0.048). DHFR rs1650697 conferred an increased risk of grade ⩾3 toxicity (OR 2.14, P=0.034). Furthermore, FOLR3 rs61734430 was associated with an increased likelihood of disease progression at mid-treatment radiological evaluation (OR 4.05, P=0.023). Polymorphisms within SLC19A1 (rs3788189, rs1051298 and rs914232) were associated with overall survival. This study confirms previous pharmacogenetic associations and identifies novel markers of pemetrexed toxicity.

Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity.

BACKGROUND: Fluoropyrimidine drugs are extensively used for the treatment of solid cancers. However, adverse drug reactions are a major clinical problem, often necessitating treatment discontinuation. The aim of this study was to identify pharmacogenetic markers predicting fluoropyrimidine toxicity. METHODS: Toxicity in the first four cycles of 5-fluorouracil or capecitabine-based chemotherapy were recorded for a series of 430 patients. The association between demographic variables, DPYD, DPYS, TYMS, MTHFR, CDA genotypes, and toxicity were analysed using logistic regression models. RESULTS: Four DPYD sequence variants (c.1905+1G>A, c.2846A>T, c.1601G>A and c.1679T>G) were found in 6% of the cohort and were significantly associated with grade 3-4 toxicity (P<0.0001). The TYMS 3'-untranslated region del/del genotype substantially increased the risk of severe toxicity (P=0.0123, odds ratio (OR)=3.08, 95% confidence interval (CI): 1.38-6.87). For patients treated with capecitabine, a MTHFR c.1298CC homozygous variant genotype predicted hand-foot syndrome (P=4.1 × 10⁻6, OR=9.99, 95% CI: 3.84-27.8). The linked CDA c.-92A>G and CDA c.-451C>T variants predicted grade 2-4 diarrhoea (P=0.0055, OR=2.3, 95% CI: 1.3-4.2 and P=0.0082, OR=2.3, 95% CI: 1.3-4.2, respectively). CONCLUSION: We have identified a panel of clinically useful pharmacogenetic markers predicting toxicity to fluoropyrimidine therapy. Dose reduction should be considered in patients carrying these sequence variants.

Effect of dietary prebiotic (mannan oligosaccharide) supplementation on the caecal bacterial community structure of turkeys.

The identification of specific bacterial species influenced by mannan oligosaccharide (MOS) supplementation may assist in the formulation of new and improved diets that promote intestinal health and improve bird performance, offering suitable alternatives to antimicrobials in feed for sustainable poultry production. This study has been conducted to evaluate the use of a MOS compound derived from the yeast cell wall of Saccharomyces cerevisiae on turkey performance, bacterial community structure and their phylogenetic associations. A 42-day turkey trial was carried out on birds fed control and MOS-supplemented diets. Bird performance data (weight gains, feed consumption and feed efficiency ratios) were collected, and caecal contents were extracted from randomly caught poults on days 28, 35 and 42 posthatch. Bird performance data showed no improvements as a result of dietary supplementation. Automated ribosomal intergenic spacer analysis (ARISA) revealed the bacterial community structure to be significantly altered on days 28 and 35 posthatch but not day 42 as a result of dietary supplementation. This technique was coupled with 16S rRNA gene sequence analysis to elucidate phylogenetic identities of bacteria. The dominant bacteria of the caecum on all days in both treatment groups were members of phylum Firmicutes, followed by the Bacteroidetes and Proteobacteria phyla, respectively. Statistical analysis of the 16S rRNA gene libraries showed that the composition of the MOS clone library differed significantly to the control on day 35 posthatch. It can be concluded that MOS alters the bacterial community structure in the turkey caecum.

An automated method for the measurement of a range of tyrosine kinase inhibitors in human plasma or serum using turbulent flow liquid chromatography-tandem mass spectrometry.

Tyrosine kinase inhibitors (TKIs) are used to treat a number of cancers, including chronic myeloid leukaemia and hepatocellular carcinoma. Therapeutic drug monitoring (TDM) may be indicated to (1) monitor adherence, (2) guide dosage, and (3) minimise the risk of drug-drug interactions and dose-related toxicity. On-line, automated sample preparation provided by TurboFlow technology (ThermoFisher Scientific) in conjunction with the sensitivity and selectivity of tandem mass spectrometry (MS/MS) detection may be applied to the analysis of single drugs and metabolites. We report the use of TurboFlow LC-MS/MS for the analysis of nine TKIs and metabolites (imatinib, N-desmethylimatinib, dasatinib, nilotinib, erlotinib, gefitinib, lapatinib, sorafenib, sunitinib) in human plasma or serum for TDM purposes. An Aria Transcend TLX-II system coupled with a TSQ Vantage was used. Samples (50 µL) were vortex mixed with internal standard solution (150 µL imatinib-D(8), gefitinib-D(8), sunitinib-D(10), and nilotinib-(13)C (2) (15) N(2) in acetonitrile) and, after centrifugation 100 µL supernatant were injected directly onto a 50 × 0.5-mm Cyclone TurboFlow column. Analytes were focussed onto a 50 × 2.1-mm (3 µm) Hypersil GOLD analytical column and eluted with an acetonitrile/water gradient. Analytes were monitored in selected reaction monitoring mode (positive APCI). Total analysis time was 7 min without multiplexing. Calibration was linear (R(2) > 0.99) for all analytes. Inter- and intra-assay precision (in percent relative standard deviation, RSD) was <11 % and accuracy 89-117 % for all analytes. No matrix effects were observed. This method is suitable for high-throughput TDM in patients undergoing chronic therapy with TKIs and has been utilised in the analysis of clinical samples.

V/Q SPECT imaging of acute pulmonary embolus - a practical perspective.

The following article is intended to illustrate the place of scintigraphy and computed tomography pulmonary angiography (CTPA) in the investigation of acute PE in current practice, and to guide non-radionuclide radiologists and other medical professionals to the best test for patients. We share our early experiences with ventilation-perfusion (V/Q) single-photon-emission computed tomography (SPECT) including image acquisition and interpretation. A comparison of the two techniques is given, along with practical considerations in a variety of clinical scenarios.

Effect of dietary supplementation with a Saccharomyces cerevisiae mannan oligosaccharide on the bacterial community structure of broiler cecal contents.

This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure.

Challenges and outcomes in establishing a sentinel lymph node biopsy service for oral squamous cell carcinoma in a regional district specialist hospital.

UK national guidelines in 2016 recommended that sentinel lymph node biopsy (SLNB) should be offered to patients with early oral squamous cell carcinoma (OSCC). We review the establishment of an OSCC SLNB service with specific consideration to resources, service implications and patient outcomes. A review of processes was performed to identify key stages in establishing the service, and subsequently a retrospective cohort study consisting of 46 consecutive patients with T1/T2 N0 OSCC was undertaken. The key stages identified were: coordinating a nuclear medicine pathway and reliable cost-appropriate pathology service, constructing a Trust business case, and gaining approval of a new interventional service policy. A median (range) of 3.3 (1-8) sentinel nodes (SLN) were removed, with 17 patients having a positive SLN. The negative predictive value of SLNB was 100%, with 12 having a SLN outside the field if elective neck dissection (END) was planned. There was a significantly increased risk of a positive SLN with increasing depth of invasion (DOI) (p=0.007) and increased diameter (p=0.036). We also identified a longer-than-ideal time to completion neck dissection and inadequate ultrasound follow up of negative SLNB patients. Establishment of a service requires careful planning. Our results were in keeping with those reported in the literature, and showed that SLNB for OSCC has a high negative predictive value and can identify at-risk SLN outside the traditional END levels, even in well-lateralised tumours. Our findings show that DOI and size of SLN were significantly associated with a positive SLN, and also identified areas requiring improvement.

Ketamine-associated lower urinary tract destruction: a new radiological challenge.

AIM: Ketamine is a short-acting dissociative anaesthetic whose hallucinogenic side effects have led to an increase in its illicit use amongst club and party goers. There is a general misconception amongst users that it is a safe drug with few long term side effects, however ketamine abuse is associated with severe urinary tract dysfunction. Presenting symptoms include urinary frequency, nocturia, dysuria, haematuria and incontinence. MATERIALS AND METHODS: We describe the radiological findings found in a series of 23 patients, all with a history of ketamine abuse, who presented with severe lower urinary tract symptoms (LUTS). Imaging techniques used included ultrasonography (US), intravenous urography (IVU), and computed tomography (CT). These examinations were reviewed to identify common imaging findings. All patients with positive imaging findings had also undergone cystoscopy and bladder wall biopsies, which confirmed the diagnosis. The patients in this series have consented to the use of their data in the ongoing research into ketamine-induced bladder pathology. RESULTS: Ultrasound demonstrated small bladder volume and wall thickening. CT revealed marked, generalized bladder wall thickening, mucosal enhancement, and perivesical inflammation. Ureteric wall thickening and enhancement were also observed. In advanced cases ureteric narrowing and strictures were identified using both CT and IVU. Correlation of clinical history, radiological and pathological findings was performed to confirm the diagnosis. CONCLUSION: This case series illustrates the harmful effects of ketamine on the urinary tract and the associated radiological findings. Delayed diagnosis can result in irreversible renal tract damage requiring surgical intervention. It is important that radiologists are aware of this emerging clinical entity as early diagnosis and treatment are essential for successful management.

Particle tracking microrheology of gel-forming amyloid fibril networks.

Microrheology is a technique that is increasingly used to investigate the local viscoelastic properties of complex fluids non-invasively, by tracking the motion of micron-sized probe spheres. In this work, passive Particle Tracking Microrheology (PTM) is used to study network formation in the milk protein beta-lactoglobulin at 80 degrees C and pH 2. In these conditions the protein aggregates to form thread-like structures known as amyloid fibrils, which can further aggregate into elastic networks. Using PTM, gels were observed to form at significantly lower concentrations than determined by bulk rheometry, where the oscillatory shear forces may disrupt either fibril or network formation. During incubation, the Mean Square Displacement (MSD) of the probe particles exhibited time-cure superposition, allowing the critical relaxation exponent to be calculated as approximately 0.63, consistent with other biopolymer gels. Combined with the gel-like appearance of the complex modulus at long incubation times, this confirms that a true gel is forming, with physical or chemical crosslinks forming between the fibrils, refining the conclusions of other workers in the field.

The use of random forests modelling to detect yeast-mannan sensitive bacterial changes in the broiler cecum.

In this study, sequencing of the 16S rRNA gene targeting the V4-V6 regions was conducted to assess the cecal microbial alterations in response to dietary supplementation with a yeast derived mannan rich fraction (MRF) in standard commercial broiler production settings across four separate broiler trials. The resulting data was analysed to identify consistent changes in the bacterial community structure of the broiler cecum in response to MRF supplementation. Subsequently, the datasets from each individual trial were pooled and analysed for differences between control and MRF supplemented diets at day 35 posthatch. The results from this analysis showed that Phylum Firmicutes was decreased and Phylum Bacteroidetes was increased across all four trials at day 35 posthatch when compared to the control. An extension of the random forest bioinformatics approach to discover a highly relevant set of microbial operational taxonomic units (OTUs) which are indicative of MRF supplementation in the broiler cecum was then used. This approach has enabled the identification of a novel set of yeast-mannan sensitive bacterial OTUs in the cecal microbiome. This information will be helpful in developing potential future nutritional strategies and will be favourable to the poultry industry.

Interleukin 2 production in vitro by peripheral lymphocytes in response to human papillomavirus-derived peptides: correlation with cervical pathology.

Human papillomavirus (HPV) is believed to be the major cause of cervical cancer. To investigate whether a cellular immune response, especially a T helper type 1 response, is related to the natural defense against HPV-related cervical lesions, the interleukin 2 response of peripheral blood lymphocytes in vitro to overlapping peptides from HPV-16 E6 and E7 oncoproteins was compared with the degree of cervical cytological abnormality among 140 women in a cross-sectional study. We compared 66 women diagnosed with low-grade squamous intraepithelial lesions (LSIL), 21 with high-grade squamous intraepithelial lesions (HSIL), and 28 with invasive cervical cancer with 25 women who were cytologically normal but previously HPV-16 DNA positive. The fraction showing strong interleukin 2 production against HPV-16 peptides was greatest among cytologically normal women (35%) and declined with increasing disease severity [LSIL] (20%), HSIL, (17%), and cancer patients (7%); X2 test P for the trend = 0.02], whereas the responses against a recall influenza antigen were not significantly different among groups. Our finding suggests that a T helper lymphocyte type 1 response to HPV antigens is associated with disease status. This result may reflect a targeted effect of the disease on immune function or a protective effect of the immune response against disease progression.

Inhibin, activin, and follistatin: regulation of follicle-stimulating hormone messenger ribonucleic acid levels.

Primary pituitary cell cultures derived from adult male rats were used to explore the direct effects of purified porcine inhibin and follistatin, and recombinant human activin A on FSH beta, as well as LH beta and alpha-subunit mRNA levels. Subunit mRNAs were determined by blot hybridization using alpha, LH beta, and FSH beta cDNA and genomic fragments. Treatment with inhibin for 72 h significantly suppressed alpha and FSH beta mRNA levels with parallel changes in FSH secretion. No change in LH beta mRNA levels was observed. A decrease in FSH beta mRNA to undetectable levels was seen 4 h after inhibin administration. Recombinant human Activin A caused dose-dependent and parallel increases in FSH beta mRNA levels and FSH secretion. This increase was evident at 4 h after activin administration and maintained at longer times. alpha and LH beta mRNA levels remained unchanged. Follistatin addition to cultures for 72 h significantly reduced FSH beta mRNA levels. In a time-course experiment, a reduction in FSH beta mRNA to undetectable levels was observed 24 h after follistatin administration. There were no changes in alpha or LH beta mRNA levels. These data demonstrate that the actions of these gonadal peptides on FSH secretion may be accounted for, at least in part at the level of biosynthesis, by reductions in FSH beta mRNA levels directly at the level of the anterior pituitary gland.

Effect of recombinant human inhibin on gonadotropin secretion by the male rat.

We have examined the effect of recombinant human inhibin-A on basal and GnRH-induced gonadotropin secretion by male rats or cultured anterior pituitary cells. Inhibin, administered sc 6 h before the experiment, induced dose- and time-related decreases in plasma FSH, but not LH, levels in both intact and castrated male rats. Inhibin also significantly interfered with the in vivo stimulatory effect of 20-500 ng GnRH on FSH release, but had inconsistent and usually modest effects on the LH response. While exposure of cultured pituitary cells to inhibin for 72 h has been reported to interfere with GnRH-induced gonadotropin release, we examined here the effects of shorter exposure periods relevant to in vivo experiments. Exposure of the cells to inhibin (31.3-312.5 pM) for 2-6 h measurably (P less than or equal to 0.01) decreased the ability of 10 nM GnRH to stimulate both FSH and LH released by cultured cells. In contrast, lower (3.1 and 9.4 pM) doses of inhibin had little or no effect. Longer exposures to inhibin (10, 24, and 72 h) increased the inhibitory effect of 31.3-312.5 pM inhibin, while 3.1 and 9.4 pM remained ineffective at all times. These results indicate that exposure of the male rat to inhibin for 6 h decreases FSH secretion, and that this effect is at least partially mediated through blunting of the pituitary response to GnRH. In contrast, the ability of inhibin to interfere with LH release, which is readily apparent in cultured pituitary cells, appears to be of lesser importance in the intact male rat.

Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells.

Activins, initially identified as FSH-releasing proteins, have now been shown to exert effects on other cell types of the anterior pituitary, including the somatotrophs. In the present study the inhibitory action of activin-A (beta A beta A) on GH secretion was characterized using primary cultures of rat anterior pituitary cells. Activin-A suppressed basal GH secretion for up to 72 h (the longest time tested). Immediately after the treatment period with activin-A, when the cells were thoroughly washed and further incubated with or without rat GH-releasing factor (rGRF), basal and stimulated GH secretion were partially inhibited as well. In parallel, activin-A pretreatment diminished rGRF-stimulated cAMP accumulation. The effects of activin-A were time- and concentration-dependent, with half-maximal inhibition occurring in the range of 20-30 pM activin-A. A minimum pretreatment time of 3 h was required for maximal effect, and when rGRF and activin-A were added simultaneously, no inhibition was evident. Secretory responses of activin-A-pretreated cells to rGRF were influenced by glucocorticoids. When cells were cultured in the presence of the synthetic glucocorticoid dexamethasone, pretreatment (72 h) with activin-A attenuated rGRF-stimulated GH secretion only during short (1-h), but not longer (3-h), exposure periods to the neuropeptide. In the absence of dexamethasone, rGRF-stimulated GH secretion was inhibited at all incubation times tested (up to 3 h). A 3-h exposure to the protein factor did not alter total (cellular plus secreted) immunoreactive GH levels, suggesting that the inhibition of secretion with the shorter treatment was not secondary to attenuated GH biosynthesis. However, longer (72-h) treatment with activin-A decreased total GH levels, indicating lower GH biosynthetic rates, as previously shown. Somatostatin is recognized as the primary negative modulator of GH secretion. Activin-A and SRIF inhibited GH secretion additively, suggesting distinct mechanisms of action for each. GH secretion in response to other secretagogues, such as 12-O-tetradecanoyl-phorbol-13-acetate, forskolin, cholera toxin, and 8-bromo-cAMP, was also suppressed after activin-A pretreatment. The presence of the RNA synthesis inhibitor actinomycin-D completely blocked the inhibitory effect of a 3-h activin-A pretreatment on subsequent rGRF-stimulated GH secretion. Pertussis toxin was only partially effective in preventing the inhibition by activin-A. The results of this study indicate that activin-A plays a crucial role as a modulator of somatotropic function, inhibiting GH secretion at the level of the secretory process and secondary to the inhibition of GH biosynthesis.

Activin stabilizes follicle-stimulating hormone-beta messenger ribonucleic acid levels.

Activin, a gonadal peptide, stimulates FSH secretion in association with an increase in FSH beta messenger RNA (mRNA) levels at the level of the anterior pituitary gland. The goal of these studies was to determine whether the effects of recombinant human activin A (rhActivin A) are exerted at the post-transcriptional level by affecting the stability of FSH beta mRNA. We determined the apparent half-life of FSH beta mRNA in the presence and absence of rhActivin A using actinomycin D. The anterior pituitary glands from adult male rats were isolated and dispersed enzymatically. Cells were preincubated in the presence of rhActivin A for 24 h to increase FSH beta mRNA levels. Actinomycin D was then added and the cells were incubated for a subsequent 4, 6, 8, 12, and 24 h in the presence or absence of rhActivin A. As reported earlier, the addition of rhActivin A caused parallel increases in FSH secretion and FSH beta mRNA levels, while having no effect on alpha or LH beta mRNA levels. Actinomycin D treatment decreased FSH beta mRNA to 49, 39, and 16% of control levels at the 4, 6, and 8 h time points, respectively. In contrast, when actinomycin D was added in the presence of rhActivin A FSH beta mRNA was reduced to 80, 58, and 42% of control levels at the 4, 6, and 8 h time points, respectively. Using the least squares method of analysis, the apparent half-lives of FSH beta mRNA under these two conditions were calculated. In the presence of actinomycin D, the half-life of FSH beta mRNA was 3.1 h. The addition of activin significantly increased the half-life to 6.5 h. These results suggest that activin A stimulates FSH beta mRNA levels, at least in part, at the posttranscriptional level by increasing the stability of FSH beta mRNA.

Activin-A regulates follistatin secretion from cultured rat anterior pituitary cells.

The activin-binding protein, follistatin (FS), was immunoprecipitated from metabolically labeled rat anterior pituitary cells or their media using a specific antiserum to purified porcine FS (anti-FS). Several immunoreactive proteins, including one that had a mobility in the range of 42-44 kilodaltons (kDa), were detected in the cell lysates. When immunoprecipitates of the culture medium were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a broad 35- to 46-kDa or 39- to 53-kDa band was visualized under unreducing or reducing conditions, respectively. Upon deglycosylation by treatment with N-glycosidase-F, the secreted product migrated as a sharp protein band with an apparent size of 35 kDa. The identity or the relatedness of the immunoprecipitated proteins to FS was verified by the ability of the C-terminally truncated form of recombinant human FS (rhFS288) to compete for binding to anti-FS. When the cultured rat anterior pituitary cells were treated with either forskolin or 12-O-tetradecanoylphorbol acetate, the accumulation of FS in the culture medium was stimulated by approximately 2.5-fold. These observations suggest that the activation of either the protein kinase A or the protein kinase C signaling pathway has a stimulatory effect on anterior pituitary FS production. A more dramatic stimulation of FS secretion (up to 7-fold) was observed when the rat anterior pituitary cells were treated with activin-A. The concentration dependence for this effect was within the same range that has been reported for most of the actions of activin-A. Inhibin-A suppressed basal FS secretion and blocked its stimulation by activin-A. To determine if locally produced FS exerts an influence on the response of gonadotropes to activins, the effects of anti-FS on FSH secretion were monitored. The ability of this FS antiserum to immunoneutralize the activity of FS was initially confirmed; anti-FS attenuated the inhibitory action of exogenous follistatin on FSH secretion. Treatment of cells with the antiserum increased the apparent sensitivity of gonadotropes to submaximal concentrations of activin-A. Moreover, the presence of the antiserum lowered the concentration of activin-A that was required to produce the maximum amount of FSH secretion, without changing the magnitude of the response. These results suggested that locally produced FS interferes with the secretory response of gonadotropes to activins. Changes in locally secreted FS may, therefore, represent a mechanism by which the response of rat anterior pituitary cells to incoming stimuli are tightly regulated.

Regulation and actions of Smad7 in the modulation of activin, inhibin, and transforming growth factor-beta signaling in anterior pituitary cells.

Activins and transforming growth factor-beta (TGF beta) are crucial autocrine, paracrine, and endocrine modulators of anterior pituitary function. Activins regulate most pituitary cells and lactotropes are targets of TGF beta. Smad2 and Smad3 are two cellular mediators of activin/TGF beta signaling, whereas Smad7 is as an inducible, negative modulator of the pathway. This study was undertaken to evaluate Smad7 regulation in the pituitary. Activin A rapidly and transiently increased Smad7 messenger RNA (mRNA) levels of rat anterior pituitary (RAP), clonal gonadotrope (alpha T 3-1 and L beta T2), and corticotrope (AtT20) cells with an EC(50) of 0.1-0.2 nM. In RAP cells, activin A or TGF beta 1 had equivalent effects that were additive. Follistatin, known to bind and inactivate activins, prevented Smad7 induction by activin. Inhibin A partially antagonized activin A, perhaps reflecting gonadotrope-selective actions. This antagonism was also evident with alpha T 3-1 and L beta T2 gonadotropes. Forskolin had no measurable effect in RAP cells, but increased Smad7 mRNA levels in alpha T3-1 cells and decreased them in L beta T2 cells. Transient transfection of Smad7 along with 3TPLux, an activin/TGF beta-responsive reporter, blocked activin-mediated promoter activation in alpha T3-1 and AtT20 cells. In alpha T3-1 cells, which express endogenous follistatin mRNA, a follistatin-luciferase reporter, rFS(rin3)-Luc, was transcriptionally activated by activin A, or when cotransfected with a constitutively active ActRIB [Alk4(T>D)], Smad2, or Smad3. Smad7 blocked rFS(rin3)-Luc activation by activin A or Alk4(T>D). Together, these results point to a role of Smad7 in modulating activin/TGF beta signaling in the pituitary.