Serum aryl hydrocarbon receptor activity is associated with survival in patients with alcohol-associated hepatitis.

BACKGROUND AND AIMS: Patients with alcohol-associated hepatitis (AH) have an altered fecal metabolome, including reduced microbiota-derived tryptophan metabolites, which function as ligands for aryl hydrocarbon receptor (AhR). The aim of this study was to assess serum AhR ligand activity in patients with AH. APPROACH AND RESULTS: The study included 74 controls without AUD, 97 patients with AUD, and 330 patients with AH from 2 different multicenter cohorts (InTeam: 134, AlcHepNet: 196). Serum AhR activity was evaluated using an AhR reporter assay with HepG2-Lucia cells incubated with serum for 24 hours. Serum AhR activity was significantly higher in patients with AH compared with both controls (1.59 vs. 0.96-fold change, p < 0.001) and patients with AUD (1.59 vs. 0.93, p < 0.001). In both AH cohorts, patients with AhR activity ≥ 2.09 had significantly lower cumulative survival rates at 30, 60, 90, and 180 days compared to those with AhR activity < 2.09. When serum AhR activity was used to further stratify patients with severe AH, the cumulative 30, 60, 90, and 180-day survival rates for patients with severe AH and the AhR activity ≥ 2.09 group were all significantly lower than those with an AhR activity < 2.09 group. CONCLUSIONS: Serum AhR activity was significantly higher in patients with AH compared with controls and individuals with AUD, and this increased activity was associated with higher mortality. Consequently, serum AhR activity holds potential as a prognostic marker.

Homeodomain Proteins SIX3 and SIX6 Regulate Gonadotrope-specific Genes During Pituitary Development.

Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific ß-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.

Gene dosage of Otx2 is important for fertility in male mice.

Together, the hypothalamus, pituitary and gonads direct the development and regulation of reproductive function in mammals. Gonadotropin-releasing hormone (GnRH) expression is limited to ∼800 neurons that originate in the olfactory placode then migrate to the hypothalamus. Coordination of the hypothalamic-pituitary-gonadal (HPG) axis is dependent upon correct neuronal migration of GnRH neurons into the hypothalamus followed by proper synthesis and pulsatile secretion of GnRH. Defects in any one of these processes causes infertility. Otx2, the vertebrate homologue of Drosophila orthodenticle, is a transcription factor that has been shown to be critical for normal brain and eye development and is expressed in both the developing GnRH neurons and the pituitary, suggesting that this gene may play a critical role in development of the HPG axis. As Otx2-null mice are embryonic lethal, we have analyzed the reproductive capacity of heterozygous Otx2 mice to determine the contribution of Otx2 gene dosage to normal HPG axis function. Our data reveal that correct dosage of Otx2 is critical for normal fertility as loss of one allele of Otx2 leads to a discernible reproductive phenotype in male mice due to disruption of the migration of GnRH neurons during development.

FOXO1 transcription factor inhibits luteinizing hormone ß gene expression in pituitary gonadotrope cells.

Synthesis of luteinizing hormone (LH) is tightly controlled by a complex network of hormonal signaling pathways that can be modulated by metabolic cues, such as insulin. One group of candidate genes that may be regulated by insulin signaling in pituitary gonadotrope cells is the FOXO subfamily of forkhead transcription factors. In this study we investigated whether FOXO1 is expressed in gonadotropes and if it can modulate LH ß-subunit (Lhb) gene expression. We demonstrated that FOXO1 is expressed in murine gonadotrope cells and that insulin signaling increased FOXO1 phosphorylation and cytoplasmic localization in a PI3K-dependent manner. We also showed that FOXO1 repressed basal transcription and gonadotropin-releasing hormone (GnRH) induction of both the murine and human LHB genes in LßT2 cells, suggesting that FOXO1 regulation of LHB transcription may be conserved between rodents and humans. Although we did not detect FOXO1 binding to the proximal Lhb promoter, the FOXO1 DNA binding domain was necessary for the suppression, suggesting that FOXO1 exerts its effect through protein-protein interactions with transcription factors/cofactors required for Lhb gene expression. FOXO1 repression mapped to the proximal Lhb promoter containing steroidogenic factor 1 (SF1), pituitary homeobox 1 (PTX1), and early growth response protein 1 (EGR1) binding elements. Additionally, FOXO1 blocked induction of the Lhb promoter with overexpressed SF1, PTX1, and EGR1, indicating that FOXO1 repression occurs via these transcription factors but not through regulation of their promoters. In summary, we demonstrate that FOXO1 phosphorylation and cellular localization is regulated by insulin signaling in gonadotropes and that FOXO1 inhibits Lhb transcription. Our study also suggests that FOXO1 may play an important role in controlling LH levels in response to metabolic cues.

Enhancement of anamnestic immunospecific antibody response in orally immunized chickens.

Production of immunospecific egg yolk antibodies (IgY antibodies) in egg laying hens through oral immunization is an attractive alternative to conventional antibody production in mammals for economic reasons as well as for animal welfare reasons. Oral immunization results in a systemic humoral response, but oral booster immunizations lack efficiency. The aim of the present study was to develop immunization schemes in which the concentration of immunospecific IgY would increase following oral booster immunizations. Two groups of egg laying hens (5 in each group) were immunized orally (each immunization event consisted of dosing on three consecutive days) with Bovine Serum Albumin (BSA) in combination with RhinoVax (RV) using different immunization schemes. A 3rd group served as a reference and received BSA emulsified in Freund's Incomplete Adjuvant (FIA) by subcutaneous injection three times and one oral dose with BSA+RV. The eggs of the chickens in this group had a significantly higher immunospecific anti BSA IgY-concentration than did any of the eggs from the orally immunized chickens. One of the immunization regimes (immunizations in weeks 1, 7 and 18) clearly included a booster effect of the immunization in week 18, demonstrating the presence of memory cells following the two initial oral immunizations. Considering that oral immunization results in approximately ten times lower concentrations of immunospecific antibodies in the egg yolk, compared to traditional subcutaneous immunization schemes, the oral immunization routines have to be further refined to compete with parenteral immunization protocols.

Egg corticosterone: a noninvasive measure of stress in egg-laying birds.

Quantitative measures of corticosteroids in biological samples that can be obtained noninvasively, such as saliva, feces, and body hair, have important potential as contributing elements in assessing the quality of captive environments and the severity of experimental procedures. Egg-laying chickens may be of particular interest because the corticosterone contents of the egg may have potential as a convenient measure of preceding adrenocortical activity. To develop methods to reliably quantify corticosterone content in the egg white and yolk, corticosterone content in eggs from 15 egg-laying chickens housed in single production cages were compared with that of eggs from 15 sister chickens, group housed in 1450 cm2 cages equipped with bedding, straw nests, sand baths, and perches. Approximately 80% of the total amount of corticosterone in the eggs was found in the yolk, and there was a positive correlation between yolk corticosterone concentration and total egg corticosterone (r = 0.90, n = 30, P < .001). The egg white contained approximately 20% of the total amount of corticosterone, but there was no correlation between concentrations in the white and the total corticosterone content of the eggs (r = 0.003). There was no difference in the white and yolk corticosterone concentrations or total egg corticosterone between singly housed and group-housed egg-laying hens. Quantitative analyses of corticosterone concentration in eggs may assist when analyzing the stressfulness of experimental procedures and major changes to the birds' environment that affect the activity of the hypothalamus-pituitary-adrenal axis.

Piezoelectric quartz crystal microbalance sensor for trace aqueous cyanide ion determination.

Using selective reaction chemistry, our present research has developed an online, real-time sensor capable of monitoring toxic cyanide at both drinking water standard and environmental regulatory concentrations. Through the use of a flow cell, aqueous samples containing cyanide are reacted with a gold electrode of a piezoelectric crystal to indirectly sense cyanide concentration by the dissolution of metallic gold. The quartz crystal is an AT-cut wafer sandwiched between two neoprene O-rings within the liquid flow cell. The presence of cyanide in solution results in the selective formation of a soluble dicyano-gold complex according to the Elsner reaction: 4Au + 8CN- + 2H2O + O2 <=> 4Au(CN)2- + 4OH-. The resulting loss of gold from the electrode is detected by the piezoelectric crystal as a resonant frequency change. Since free cyanide is a weak acid (pKa = 9.3), available protons compete for cyanide ligands. Therefore, increased sample pH provides higher sensitivity. The detection limits at pH 12 are 16.1 and 2.7 ppb for analysis times of 10 min and 1 h, respectively. The incorporation of the flow cell improves both analyte sensitivity and instrument precision, with an average signal intensity drift of only 5% over a 2-h analysis. The calibrations show excellent linearity over a variety of cyanide concentrations ranging from low ppb to hundreds of ppm. This detection method offers the advantage of selectively detecting cyanides posing a biohazard while avoiding detection of stable metal cyanides. This aspect of the system is based on competitive exchange of available metals and gold with cyanide ligands. Stable metal cyanide complexes possess a higher formation constant than cyanoaurate. This detection system has been configured into a flow injection analysis array for simple adaptation to automation. Anions commonly found in natural waters have been examined for interference effects. Additionally, the sensor is free from interference by aqueous cyanide analogues including thiocyanate. The developed detection system provides rapid cyanide determinations with little sample preparation or instrument supervision.

Correlation between adjuvanticity and immunogenicity of cholera toxin B subunit in orally immunised young chickens.

The aim of the present study was to investigate whether the adjuvanticity of the cholera toxin B (CTB) subunit was correlated with its immunogenicity in young orally immunised chickens. Thirteen 15-day-old chickens were orally immunised with bovine serum albumin (BSA) glutaraldehyde coupled to CTB. The chicken antibody (IgG) concentrations against BSA and CTB, respectively, were quantified by ELISA. A significant positive correlation (r=0.66, n=39, p<0.001) between the concentrations of immunospecific antibodies with specificities against BSA and CTB, respectively, demonstrated that the adjuvanticity of CTB is correlated with its immunogenicity.

RhinoVax is an efficient adjuvant in oral immunisation of young chickens and cholera toxin B is an effective oral primer in subcutaneous immunisation with Freund's incomplete adjuvant.

Forty-five approximately 50% in-bred 14-day-old White Leghorn female chickens (Gallus domesticus) originating from 11 hens were distributed into 5 treatment groups containing one sister in each treatment group. Phase I involved oral administration of an antigen, Bovine Serum Albumin (BSA), in combination with various adjuvant preparations, either Cholera Toxin B-subunit (CTB) and/or RhinoVax (RV). A positive control group received BSA emulsified in Freund's Incomplete Adjuvant (FIA) by subcutaneous injection. All chickens responded with immunospecific IgA, IgM and IgG antibodies in their circulation. Classical parenteral immunisation with FIA was generally the most potent mode of antigen administration. The highest immunospecific IgG concentrations recorded in the orally-immunised chickens were in the group immunised with 20% RV as the adjuvant. The concentration in this group was approximately 5 times lower than that recorded in the FIA group. For practical egg yolk polyclonal antibody production purposes, the oral regime using 20% RV as adjuvant seems an attractive alternative to the more invasive technique of injecting the antigen in FIA emulsions. In Phase 2 all chickens were subjected to traditional subcutaneous immunisation with a new antigen, human IgG emulsified in FIA. The two groups of chickens that had received CTB orally during Phase I responded with significantly higher immunospecific antibody concentrations than did the other chickens, indicating that oral administration of CTB prior to traditional parenteral immunisation may have a priming effect on the humoral immune system. The immunospecific antibody response varied between the 11 families of chickens. There was no correlation between familial responsiveness to oral and subcutaneous immunisations. Families that were high responders to oral immunisation were not high responders to parenteral immunisation and vice versa.

Molecular analysis of Clostridium difficile strains isolated from 18 cases of recurrent clostridium difficile-associated diarrhea.

Recurrence of Clostridium difficile-associated diarrhea (CDAD) occurs in 15 to 20% of patients after discontinuation of treatment. Arbitrarily primed PCR was used to investigate the epidemiology of recurrent CDAD in 18 patients. Reinfection with a new strain occurred in 6 of 18 patients (33.3%), while 12 patients relapsed with the original strain shortly after discontinuation of treatment. These data suggest that reinfection with exogenous C. difficile is a common problem and that not all recurrences are due to relapse.

Hemocyte production and maturation in an invertebrate animal; proliferation and gene expression in hematopoietic stem cells of Pacifastacus leniusculus.

Regulation of hematopoiesis in invertebrates is largely unknown, although the hemocytes are essential in immunity, performing functions such as phagocytosis, encapsulation and lysis of foreign cells. We have developed a method to isolate hematopoietic stem cells from the freshwater crayfish, Pacifastacus leniusculus, and therefore, this animal provides a powerful tool for studies on invertebrate hematopoiesis. The hematopoietic tissue of crayfish was found to be actively proliferating. Injection of a beta1,3-glucan caused a severe loss of hemocytes, followed by a rapid recovery, due to release from the hematopoietic organ. Transcripts for peroxinectin, a hemocyte cell adhesion protein, were present in the hematopoietic cells, whereas mRNA for proPO was not detected. A gene coding for a Runt-domain protein known to be involved in hematopoiesis in Drosophila and mammals, was upregulated prior to hemocyte release.We conclude that hemocytes are synthesised and partly differentiated in the hematopoietic tissue, but the final differentiation into functional hemocytes expressing proPO is not completed until the hemocytes are released into the circulation.