Lipopolysaccharide-induced TNF Research Articles

Bacillus subtilis inhibits intestinal inflammation and oxidative stress by regulating gut flora and related metabolites in laying hens

Bacillus subtilis is one of the most popular commercial probiotics used in farm animal production. However, its potential mechanisms are not very clear. The aim of this study was to investigate the effects of dietary Bacillus subtilis on intestinal histomorphology, innate immunity, microbiota composition, transcriptomics, and related metabolomics. Twenty-four 48-week-old Lohman Pink-shell laying hens were randomly divided into two groups: a basic diet and the basic diet supplemented with Bacillus subtilis (0.5 g/kg) for a 9-week experiment. At the end of the experiment, tissues of the duodenum, ileum, and jejunum as well as cecal content of each bird were collected for microstructure, PCR, transcriptome, metabolome, and 16S rRNA analyses. The results showed that dietary Bacillus subtilis supplement had no effect on the intestinal microstructure. However, Bacillus subtilis increased mRNA expression of tight junction protein occludin (P < 0.05), while reduced mRNA expression of lipopolysaccharide-induced TNF factor (P < 0.01) in the duodenum. Moreover, transcriptomic results indicated that most of Bacillus subtilis supplement-induced differential genes were associated with inflammation and immunity, including cytochrome b-245 beta chain, transferrin, and purinergic receptor P2X 7, resulting in a decrease in Malondialdehyde level (P < 0.05) in the duodenum. In addition, at the genus level, Bacillus subtilis supplement enriched the potential beneficial bacteria, Candidatus_Soleaferrea (P = 0.02) but inhibited the harmful bacteria including Lachnospiraceae_FCS020_group, Ruminiclostridium, Lachnospiraceae_UCG-010, and Oxalobacter. Metabolomic results revealed that N-Acetylneuraminic acid and ADP were increased by fed Bacillus subtilis. These results suggest that dietary Bacillus subtilis could inhibit gut inflammation and improve antioxidative status and barrier integrity of the duodenum via regulating gut microbial composition in laying hens.

Effects of zinc glycinate on growth, immunity, and intestinal health in broiler chickens

The objective is to investigate the effects of dietary zinc form and concentration in broiler chickens. Day-old broiler chicks (n = 6) were allotted to treatments in a 2 × 3 factorial arrangement to examine supplemental zinc from sulfate (ZS) and glycinate (ZG) and concentration (40, 80, and 120 mg/kg). In healthy conditions, no difference in poultry performances is to be expected between organic and inorganic zinc sources, supplemented at the same zinc dose to the diets. We found that on day 21, zinc supplemented at 80 mg/kg increased body weight gain (BWG) by 113 g compared to zinc supplemented at 40 mg/kg (P < 0.01). Similarly, on day 35, zinc supplemented at 120 mg/kg increased BWG by 155 g compared to zinc supplemented at 40 mg/kg (P < 0.05). On day 35, zinc supplemented at 120 mg/kg increased feed intake by 239 g compared to zinc supplemented at 40 mg/kg (P < 0.05). The villi length on day 21 was increased by 63 µm in birds supplemented at 120 mg/kg compared to 40 mg/kg (P < 0.05). On day 21, cecal tonsil gene expression of interleukin 1 beta (IL-1β) was higher by 2.5 and 2.4-fold in the ZG-80 mg/kg group than the ZG-40 mg/kg and ZS-120 mg/kg groups (P < 0.01), and interleukin 10 (IL-10) was lower by 2-fold in the ZS-120 mg/kg group than ZS-40 mg/kg, while IL-10 was lower in the ZG-40 mg/kg than ZS-40 mg/kg group (P < 0.01). On day 35, cecal tonsil gene expression of IL-1β was overall higher by 1.3-fold in the ZG group compared to ZS (P < 0.05). On day 21, spleen gene expression of transforming growth factor-beta (TGF-β) was higher by 1.4-fold in the ZG-80 mg/kg group compared to ZG-40 mg/kg (P < 0.05). On day 35 spleen gene expression of lipopolysaccharide-induced TNF factor (LITAF) was lower in the ZG-80 mg/kg group compared to ZS-80 mg/kg (P < 0.05). On day 35 jejunum gene expression of claudin-2 was higher by 2.1-fold in the ZG-80 mg/kg group than ZS-80 mg/kg (P < 0.05). This study supports that zinc supplementation at 120 mg/kg improves the growth performance of broiler chickens. This is accompanied by improvements in intestinal morphology and modulation in immunity through an altered cytokine production profile.

Sambucus nigra: A traditional medicine effective in reducing inflammation in mice

Ethnopharmacological relevanceSambucus nigra L. is a plant of European origin and popularly known as elder, elderberry, black elder, European elder, European elderberry, and European black elderberry, being described in pharmacopoeia of several countries. Its flowers and berries have been used in folk medicine to treat feverish conditions, coughing, nasal congestion, and influenza besides its popular use as anti-inflammatory, analgesic, and diuretic agent. Aim of the studyThe aim of this investigation was to elucidate the anti-inflammatory and the relaxant effect of the lyophilized aqueous extract obtained from S. nigra's flowers on in vivo and in vitro inflammation assays and on the isolated rat vascular and airway smooth muscle tissue. Material and methodsThe anti-inflammatory activity of the extract was investigated using carrageenan-induced inflammation model in the subcutaneous tissue of male Swiss mice orally treated with S. nigra extract (30, 100, 300 or 600 mg/kg). Leukocyte influx and the secretion of chemical mediators were quantified in the inflamed exudate. Additionally, histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1β, IL-10 and NO production, and adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry) were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with LPS and treated with the extract (1, 10 or 100 μg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. Finally, the effect of the extract was evaluated in isolated rat aorta and trachea rings. ResultsThe oral treatment with S. nigra promoted reduction in the neutrophil migration as well as the decrease of TNF, IL-1β and IL-6 levels in the inflamed exudate. In vitro treatment with S. nigra decreased NO2−, TNF, IL-1β and IL-6 and promoted increase of IL-10 in LPS-stimulated neutrophils. Similarly, the extract reduced the NO2−, TNF and IL-6 in LPS-stimulated macrophages. Rutin, the major constituent of S. nigra extract reduced NO2−, TNF, IL-1β, and IL-6 and promoted the increase of IL-10 in LPS-stimulated neutrophils supernatant. The extract also shed CD62L and CD18 expressions. The extract was able to increase the efferocytosis of apoptotic neutrophils by increasing the IL-10 and decreasing the TNF levels. Additionally, the extract reduced the hypersensitivity induced by carrageenan and promoted a relaxant effect in isolated vascular and non-vascular rat tissue. ConclusionsS. nigra flowers extract presents anti-inflammatory effect by modulating macrophage and neutrophil functions including the production of inflammatory mediators and cell migration, by promoting efferocytosis and consequently the resolution of acute inflammation, besides exerting antinociceptive effects, scientifically proving its popular use as medicinal plant. Allied to the relaxant effect in both vascular and non-vascular smooth muscle tissue, S. nigra extract represents an important tool for the management of acute inflammation.

Study on the interactive effect of deoxynivalenol and Clostridium perfringens on the jejunal health of broiler chickens

With global warming and ban on antibiotics, it occurs occasionally that deoxynivalenol (DON) together with Clostridium perfringens impairs the gut health of broiler chickens. However, the interactive effect of DON and C. perfringens on intestinal health is still unknown. A total of 120 one-day-old Arbor Acres broilers were randomly distributed to 4 groups. Birds were gavaged with C. perfringens (8 × 108 CFU/d per bird) or sterile medium and fed a DON diet (0 or 5 mg of DON per kg diet) to investigate the interactive effects. The main effect analysis showed that DON diet significantly downregulated (P < 0.05) the mRNA expression of mucin-2, B-cell lymphoma-2–associated X, and cysteinyl aspartate–specific proteinase-3 of jejunal mucosa; decreased (P < 0.05) the indexes of ACE, Chao1, Shannon, and Simpson; and also decreased the relative abundance of the phylum Bacteroidete and the genera Lactococcus in jejunal contents of broilers chickens. Meanwhile, C. perfringens significantly increased (P < 0.05) crypt depth; decreased (P < 0.05) the ratio of villi height to crypt depth, the activity of jejunal diamine oxidase, and the relative abundance of Lactococcus; and upregulated (P < 0.05) the relative expression of B-cell lymphoma-2 and cysteinyl aspartate–specific proteinase-8. Furthermore, the interactions between DON and C. perfringens were most significant (P < 0.05) in the mRNA expression of lipopolysaccharide-induced TNF factor (LITAF) and TLR-4, the abundance of the genera Lactococcus in jejunal contents, and butyric acid concentrations in cecal contents of birds. Finally, Spearman correlation analysis suggested that the most negative correlations (P < 0.05) with the abundance of the genera except Lactobacillus were observed within the mRNA expression of LITAF. The abundance of Lactococcus had a positive correlation (P < 0.05) with the expression of Caspase-3. Most genera except Lactobacillus negatively correlated (P < 0.05) with acetic acid, butyric acid, and total short-chain fatty acids. In conclusion, dietary deoxynivalenol and C. perfringens challenge had a harmful effect on the jejunal health and should be carefully monitored in broiler production.

Effects of Tithonia diversifolia (Asteraceae) extract on innate inflammatory responses

Ethnopharmacological relevanceTithonia diversifolia (Helms.) A. Gray, popularly known in Brazil as “margaridão” or “mão-de-Deus” has been used in the folk medicine as anti-inflammatory and against other illnesses in several countries. Indeed, many studies show de effect of T. diversifolia in the inflammatory process, however, any of them have demonstrated the mechanism of cell migration. Aim of the studyThe aim of this investigation was to show the in vivo and in vitro effects of T. diversifolia leaves ethanol extract on neutrophil trafficking from the blood to the inflamed tissue and on cell-derived secretion of chemical mediators, as well as, the effects on inflammatory resolution and inflammatory pain. Materials and methodsAnti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally treated with the T. diversifolia extract (0.1, 1 or 3 mg/kg). The leukocyte influx (optical microscopy) and the secretion of chemical mediators (TNF, IL-6, IL-1β and CXCL1, by enzyme-linked immunosorbent assay) were quantified in the inflamed exudate. Histological analysis of the pouches was performed. N-Formyl-methionine-leucine-phenylalanine-induced chemotaxis, lipopolysaccharide-induced TNF, IL-6, IL-1β, CXCL1 and NO production, and adhesion molecule expression (CD62L and CD18, flow cytometry) were in vitro quantified using oyster glycogen recruited peritoneal neutrophils previous treated with the extract (1, 10, or 100 μg/mL). The resolution of inflammation was accessed by efferocytosis assay, and the antinociceptive activity was investigated using carrageenan-induced mechanical hypersensitivity. ResultsThe oral treatment with T. diversifolia promoted reduction in the neutrophil migration as well as the decrease in total protein, TNF, IL-1β and CXCL1 levels in the inflamed exudate. In vitro treatment with T. diversifolia shedding of β2 integrin expressions, without alter CD62L expression. The extract was able to increase the efferocytosis of apoptotic neutrophils, and the increase of the IL-10 and the decrease of TNF secretion. Additionally, the extract reduced the hypersensitivity induced by carrageenan. ConclusionsTogether, the data herein obtained showed that T. diversifolia extract presented anti-inflammatory activity by inhibiting the cytokine and NO production, and also the leukocyte migration. The mechanisms involved in the extract anti-inflammatory effects include the impairment in the leukocyte migration to the inflamed tissue, the pro-resolution activity, and consequently the anti-hypersensitivity.

IRF5 Is a Key Regulator of Macrophage Response to Lipopolysaccharide in Newborns.

Infections are a leading cause of mortality and morbidity in newborns. The high susceptibility of newborns to infection has been associated with a limited capacity to mount protective immune responses. Monocytes and macrophages are involved in the initiation, amplification, and termination of immune responses. Depending on cues received from their environment, monocytes differentiate into M1 or M2 macrophages with proinflammatory or anti-inflammatory and tissue repair properties, respectively. The purpose of this study was to characterize differences in monocyte to macrophage differentiation and polarization between newborns and adults. Monocytes from umbilical cord blood of healthy term newborns and from peripheral blood of adult healthy subjects were exposed to GM-CSF or M-CSF to induce M1 or M2 macrophages. Newborn monocytes differentiated into M1 and M2 macrophages with similar morphology and expression of differentiation/polarization markers as adult monocytes, with the exception of CD163 that was expressed at sevenfold higher levels in newborn compared to adult M1 macrophages. Upon TLR4 stimulation, newborn M1 macrophages produced threefold to sixfold lower levels of TNF than adult macrophages, while production of IL-1-β, IL-6, IL-8, IL-10, and IL-23 was at similar levels as in adults. Nuclear levels of IRF5, a transcription factor involved in M1 polarization, were markedly reduced in newborns, whereas the NF-κB and MAP kinase pathways were not altered. In line with a functional role for IRF5, adenoviral-mediated IRF5 overexpression in newborn M1 macrophages restored lipopolysaccharide-induced TNF production. Altogether, these data highlight a distinct immune response of newborn macrophages and identify IRF5 as a key regulator of macrophage TNF response in newborns.

Modulation of cytokine gene expression by selected Lactobacillus isolates in the ileum, caecal tonsils and spleen of Salmonella-challenged broilers

Probiotics have been used to control Salmonella colonization in the chicken intestine. Recently, we demonstrated that certain selected Lactobacillus isolates were able to reduce Salmonella infection in the chicken spleen and liver as well as down-regulated Salmonella pathogenicity island 1 virulence gene expression in the chicken caecum. To further understand the mechanisms through which Lactobacillus protected chickens from Salmonella infection, the present study has investigated the Lactobacillus isolate(s)-induced host immune response of chickens to Salmonella enterica serovar Typhimurium infection. A thorough examination of cytokine gene expression in the ileum, caecal tonsils, and spleen on days 1 and 3 post-Salmonella infection showed a dynamic spatial and temporal response to Salmonella infection and Lactobacillus treatments. In most instances, it was evident that treatment of chickens with Lactobacillus isolates could significantly attenuate Salmonella-induced changes in the gene expression profile. These included the genes encoding pro-inflammatory cytokines [lipopolysaccharide-induced TNF factor, interleukin (IL)-6, and IL-8], T helper 1 cytokines [IL-12 and interferon (IFN)-γ], and T helper 2 cytokines (IL-4 and IL-10). Another important observation from the present investigation was that the response induced by a combination of Lactobacillus isolates was generally more effective than that induced by a single Lactobacillus isolate. Our results show that administration of certain selected Lactobacillus isolates can effectively modulate Salmonella-induced cytokine gene expression, and thus help reduce Salmonella infection in chickens.

A Simple Regulation of Cardiomyocyte Excitability

Introduction: Recently, genome wide association studies (GWAS) identified LITAF (lipopolysaccharide-induced TNF factor) as one of the novel loci associated with prolongation of QT interval. Our goal is to investigate the role of LITAF in regulating cardiomyocyte excitability and action potential duration (APD). Methods: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients and transmembrane (TM) potentials. Propagation velocities of depolarizing waves were determined. Action potential (AP) was digitally imaged in adenoviral infected neonatal rabbit cardiomyocytes (NRbCM). Live cell confocal calcium imaging was performed on adult rabbit ventricular myocytes (ARbCM) to determine intracellular Ca2+ handling. A current-voltage (I-V) relationship for the L-type calcium current (ICa,L ) was determined using whole cell patch clamping. To study the effect of LITAF on Cav1.2 channel expression, surface biotinylation and western blot assays were performed using tSA201 cells. Results: Our studies demonstrate that loss-of-function of LITAF in vivo (zebrafish) increases the Ca2+ transient amplitude (which primarily depends on calcium influx), slows atrial-ventricular conduction, and prolongs the APD. In contrast, in vitro gain-of-function experiments in rabbit CM established that overexpression of LITAF downregulates the total and surface pools of Cav1.2 channel polypeptides, decreases Ca2+ transients and L-type Ca2+ conductance, and shorten the APD. Studies in heterologous expression systems revealed that one of the auxiliary subunits of Cav1.2 channels, Cavβ2b, is likely involved in LITAF-mediated inhibition of recombinant Cav1.2 channels. Conclusion: LITAF regulates the expression and function of Cav1.2 channels, thereby determining APD and cardiac cell excitation.

Gene expression profiling in adipose tissue from growing broiler chickens

In this study, total RNA was collected from abdominal adipose tissue samples obtained from ten broiler chickens at 3, 4, 5, and 6 weeks of age and prepared for gene microarray analysis with Affymetrix GeneChip Chicken Genome Arrays (Affymetrix) and quantitative real-time PCR analysis. Studies of global gene expression in chicken adipose tissue were initiated since such studies in many animal species show that adipose tissue expresses and secretes many factors that can influence growth and physiology. Microarray results indicated 333 differentially expressed adipose tissue genes between 3 and 6 wk, 265 differentially expressed genes between 4 and 6 wk and 42 differentially expressed genes between 3 and 4 wk. Enrichment scores of Gene Ontology Biological Process categories indicated strong age upregulation of genes involved in the immune system response. In addition to microarray analysis, quantitative real-time PCR analysis was used to confirm the influence of age on the expression of adipose tissue CC chemokine ligands (CCL), toll-like receptor (TLR)-2, lipopolysaccharide-induced TNF factor (LITAF), chemokine (C-C motif) receptor 8 (CCR8), and several other genes. Between 3 and 6 wk of age CCL5, CCL1, and CCR8 expression increased (P = 0.0001) with age. Furthermore, TLR2, CCL19, and LITAF expression increased between 4 and 6 wk of age (P = 0.001). This is the first demonstration of age related changes in CCL, LITAF, and TLR2 gene expression in chicken adipose tissue. Future studies are needed to elucidate the role of these adipose tissue genes in growth and the immune system.

Morphine Prevents Lipopolysaccharide-Induced TNF Secretion in Mast Cells Blocking IκB Kinase Activation and SNAP-23 Phosphorylation: Correlation with the Formation of a β-Arrestin/TRAF6 Complex

We have previously shown that morphine pretreatment inhibits mast cell-dependent TNF production after LPS injection in the murine peritoneal cavity. In this study, we used bone marrow-derived mast cells (BMMCs) to investigate the molecular mechanisms of that inhibition. We found that morphine prevented LPS-induced TNF secretion in these cells. The observed inhibition was not due to morphine-induced TLR4 internalization and it was related to the blockage of preformed TNF secretion. LPS-induced TNF exocytosis in BMMCs was dependent on tetanus toxin-insensitive vesicle-associated membrane proteins and calcium mobilization, as well as PI3K, MAPK, and IκB kinase (IKK) activation. TNF secretion was also associated to the phosphorylation of synaptosomal-associated protein 23 (SNAP-23), which was found forming a complex with IKK in LPS-activated BMMCs. Morphine pretreatment prevented TLR4-dependent ERK and IKK phosphorylation. Analyzing the signaling events upstream of IKK activation, we found diminished TGF-β-activated kinase 1 (TAK1) phosphorylation and TNFR-associated factor (TRAF) 6 ubiquitination in BMMCs pretreated with morphine and stimulated with LPS. Morphine pretreatment provoked a marked increase in the formation of a molecular complex composed of TRAF6 and β-arrestin-2. Naloxone and a combination of μ and δ opioid receptor antagonists prevented morphine inhibitory actions. In conclusion, our results show that activation of μ and δ opioid receptors with morphine suppresses TLR4-induced TNF release in mast cells, preventing the IKK-dependent phosphorylation of SNAP-23, which is necessary for TNF exocytosis, and this inhibition correlates with the formation of a β-arrestin-2/TRAF6 complex. To our knowledge, these findings constitute the first evidence of molecular crosstalk between opioid receptors and the TLR4 signal transduction system in mast cells.

A Novel Leflunomide Analog, UTL-5b (GBL-5b), Suppresses JAK3, MAP3K2, and LITAF Genes

UTL-5b (GBL-5b) is a novel analog of leflunomide with anti-inflammatory and antiarthritic effects. It has been shown to lower serum tumor necrosis factor-alpha (TNF-α) level induced by lipopolysaccharide (LPS) in an animal model. In this study, the effect of UTL-5b on nitric oxide (NO) and dihydroorotate dehydrogenase (DHODH) was investigated. Our in vitro studies showed that (1) UTL-5b is a stronger inhibitor of NO production as compared to leflunomide and its active metabolite, teriflunomide, and (2) Unlike leflunomide, a potent inhibitor of DHODH, UTL-5b does not inhibit DHODH activity. These findings show that UTL-5b acts in a manner different from that of leflunomide. To further investigate the mode of action of UTL-5b, an ex vivo gene array study was performed. C57BL/6 mice were injected subcutaneously with of UTL-5b 24 hr before injection of E. coli LPS. Mice were sacrificed 90 min later and the whole spleen mRNA was isolated for gene microarray analysis. The results showed that UTL-5b significantly suppressed three genes that are relevant to the TNF- pathway: Janus kinase 3 (JAK3), mitogen-activated protein kinase kinase kinase 2 (MAP3K2) and lipopolysaccharide-induced TNF- factor (LITAF). In summary, our results showed that UTL-5b has a stronger inhibitory effect on NO production than leflunomide; yet, unlike leflunomide, UTL-5b does not inhibit DHODH in vitro. In addition, gene array analysis showed that the biological effects of UTL-5b are attributed at least in part to the suppression of JAK3, MAP3K2 and LITAF gene expression.

Identification of Human Gene Products Containing Pro-Pro-x-Tyr (PY) Motifs that Enhance Glutathione and Endocytotic Marker Uptake in Yeast

In an attempt to identify genes involved in glutathione (GSH) transport, a human mammary gland cDNA library was screened for clones capable of complementing a defect in GSH uptake in yeast cells that lack Hgt1p, the primary yeast GSH uptake transporter. Five genes capable of rescuing growth on sulfur-deficient GSH-containing medium were identified: prostate transmembrane protein, androgen induced 1 (PMEPA1); lysosomal-associated protein transmembrane 4 alpha (LAPTM4α); solute carrier family 25, member 1 (SLC25A1); lipopolysaccharide-induced TNF factor (LITAF); and cysteine/tyrosine-rich-1 (CYYR1). All of these genes encode small integral membrane proteins of unknown function, although none appear to encode prototypical GSH transporters. Nevertheless, they all increased both intracellular glutathione levels and [³H]GSH uptake rates. [³H]GSH uptake was uniformly inhibited by high concentrations of unlabeled GSH, GSSG, and ophthalmic acid. Interestingly, each protein is predicted to contain Pro-Pro-x-Tyr (PY) motifs, which are thought to be important for regulating protein cell surface expression. Uptake of the endocytotic markers lucifer yellow and FM4-64 was also enhanced by each of the five genes. Mutations of the PY motifs in LITAF largely abolished all of its effects. In summary, although the results do not reveal novel GSH transporters, they identify five PY-containing human gene products that may influence plasma membrane transport activity.

Disruption of the Salmon Reproductive Endocrine Axis Through Prolonged Nutritional Stress: Changes in Circulating Hormone Levels and Identification of Differentially Expressed Ovarian Genes.

Environmental stressors (e.g., reduced food availability, hypoxia, endocrine disrupting chemicals) are known to disrupt endocrine function of fish, potentially delaying the age of puberty and affecting gamete quality. Assessing the reproductive health of fish in early stages of gametogenesis using histological and hormonal indices provides useful information, but has limited prognostic value for future reproductive potential. Our long-term aim is to develop tools for monitoring environmental impacts on ovarian growth in fish using large-scale ovarian gene expression analyses. However, little is known about the regulation of gonadal gene expression during previtellogenic oocyte growth in either healthy growing fish or fish exposed to environmental conditions that have been shown to disrupt the reproductive endocrine system and inhibit gonadal growth. The goal of this study was to identify genes that are specifically up- or down-regulated in the previtellogenic ovary of coho salmon when ovarian growth is impaired by prolonged fasting. Two year-old fish were individually tagged and fasted over a 17-week period, which resulted in less than 20% loss of body weight. Prolonged fasting caused regression of the ovaries and disrupted the reproductive endocrine axis, as fasted salmon showed significantly lower gonad weights, oocyte diameters, and plasma estradiol-17β (E2) levels relative to fed animals by week 14. Further, a higher proportion of atretic oocytes was observed in ovaries of fasting fish at week 14 and numbers increased by week 17. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries using ovaries from fed and fasted animals at week 14 were generated. A total of 480 clones were sequenced per library and their putative identity determined by comparing sequences to the NCBI and fish EST databases. The library representing genes up-regulated in the fed ovary included steroidogenesis related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), P450-aromatase (cyp19a1a), and TGF-β superfamily members, such as anti-Mullerian hormone (amh) and inhibin (inha). The library representing genes up-regulated in the fasted ovary included genes that have been associated with apoptosis, such as programmed cell death protein 4 (pdcd4), lipopolysaccharide-induced TNF factor (litaf), cullin 3 (cul3), and kruppel-like factor 6 (klf6). The genes described above were examined with quantitative PCR and results confirmed that the SSH libraries were enriched in transcripts differentially expressed between ovaries of fed and fasted salmon. This study has revealed ovarian genes that are important to normal ovarian development and those that are affected by nutritional stress and may relate to apoptosis/atresia within the ovary. Ultimately, this and subsequent studies should provide new tools that will enable molecular based assessment of fish reproductive health. This research was funded by Washington Sea Grant project number R/B-49. (poster)

Changes in the expression of interleukin-1β and lipopolysaccharide-induced TNF factor in the oviduct of laying hens in response to artificial insemination

The aim of this study was to determine the physiological significance of interleukin-1beta (IL1B) and lipopolysaccharide-induced TNF factor (LITAF) in the fate of sperm in the oviduct of laying hens after artificial insemination (AI). Laying hens were inseminated with fresh semen, PBS or seminal plasma and tissues from different oviductal segments were collected to observe the general histology, changes in the mRNA expression of IL1B and LITAF and the localization of positive cells expressing immunoreactive IL1B (irIL1B). Semi-quantitative RT-PCR was used to observe the changes in mRNA expression of these molecules in the infundibulum, uterus, utero-vaginal junction (UVJ), and vagina after insemination. Intact sperm in the lumen and between the primary or secondary folds of the vagina were found until 6 h after insemination but were degraded at 12 h. The mRNA expression of IL1B and LITAF was significantly increased in the vagina until 6 h after AI but remained unchanged in the other oviductal segments. In the tissue of the vagina and UVJ, irIL1B was localized in the mucosal stroma. The number of irIL1B-positive cells was increased in the vagina but almost unchanged in UVJ after insemination with semen. Significant changes were not observed in the mRNA expression and irIL1B-positive cells in the vagina after PBS or seminal plasma insemination. The increase of IL1B and LITAF in the vagina may lead to sperm degradation and elimination by cilia of surface epithelium, whereas their lower levels in UVJ may permit sperm to survive in sperm storage tubules.

A mechanistic approach to understanding conjugated linoleic acid's role in inflammation using murine models of rheumatoid arthritis

A naturally occurring fatty acid, conjugated linoleic acid (CLA), reduces immune-induced TNF and inducible cyclooxygenase (COX-2) expression; key mediators of inflammation in rheumatoid arthritis (RA). On the basis of previous work, it was hypothesized that dietary CLA would act as an anti-inflammatory agent in select animal models of RA. In the collagen antibody-induced arthritis (CAIA) model, mice fed CLA (mixed isomers of c9, t11, and t10, c12-CLA) for 3 wk before anticollagen antibody injection had reduced lipopolysaccharide-induced plasma TNF levels and had arthritic scores that were 60% of mice fed corn oil (CO). In the collagen-induced arthritis (CIA) model, mice fed mixed isomers of CLA for 21 days before immunization had lower IgG(1) titers, earlier signs of joint inflammation, but similar arthritis scores compared with CO fed mice during the remaining 70-day post-injection period. Beginning on day 80 to 133, CLA-fed mice had arthritic scores 70% that of the CO-fed mice. In a second CIA experiment, CLA was fed only after the booster injection. Plasma IgG(1) levels were not reduced and arthritis onset was delayed 4 days in CLA-fed mice compared with the CO-fed mice. Peak arthritis score was similar between CLA and CO-fed mice from day 35 to 56. Because CLA reduced inflammation in the CAIA model, delayed onset of arthritis in the CIA model (CIA experiment 2) and reduced arthritis score after day 80 in the CIA model (CIA experiment 1), we concluded that dietary CLA exhibited anti-inflammatory activity that was dependent on antibody.

Molecular cloning and characterization of chicken lipopolysaccharide-induced TNF- α factor (LITAF)

The inflammatory response to parasites, bacteria, and viruses is mediated by multiple host factors. TNF- α is one of the most pleiotropic cytokines in mammals, but has yet to be identified in avian species. In the current study, we isolated a full-length cDNA encoding the chicken homologue of LPS-induced TNF- α factor (LITAF), transcription factor, with an open reading frame of 148 amino acids and a predicted molecular mass of 16.0 kDa. Quantitative RT-PCR analysis showed that chicken LITAF mRNA was predominantly expressed in spleen and intestinal intraepithelial lymphocytes (IELs). LITAF mRNA levels were up-regulated following in vitro stimulation of macrophages for 4 h with Escherichia coli or Salmonella typhimurium endotoxin, and 18–48 h after treatment with Eimeria acervulina, E. maxima, or E. tenella, three causative agents of avian coccidiosis. LITAF mRNA was up-regulated by more than 700-fold in IELs isolated from E. maxima-infected birds. Purified recombinant LITAF protein expressed in E. coli or COS7 cells exhibited cytotoxic activity against chicken tumor cell lines in vitro, presumably through autocrine activation of TNF- α or its chicken homologue. This supposition was strengthened by the fact that treatment of chicken macrophages with recombinant LITAF induced the expression of TL1A (TNFSF 15), a member of the TNF ligand super family. We conclude that chicken LITAF may play an important role in the regulation of TNF- α gene expression during the course of coccidiosis or tumorigenesis.

Adenovirus Infection Dramatically Augments Lipopolysaccharide-Induced TNF Production and Sensitizes to Lethal Shock

We observed a remarkable synergism of adenoviruses and LPS in triggering the production of TNF in intact animals. We found that in mice pre-exposed to adenoviruses, LPS injections generated extremely high levels of TNF with altered kinetics. The elevated TNF synthesis stemmed mostly from posttranscriptional up-regulation of TNF production, although transcription of the TNF gene was also induced. Adenoviruses and LPS exhibited a significant but less dramatic synergism in the induction of IL-6, IFN-gamma, and NO. Only marginal changes were detected in the synthesis of a panel of other cytokines. Different serotypes of the virus showed practically identical effects. As deletion mutants lacking indispensable viral genes or UV inactivated virions exhibited similar activities as the infectious, wild-type virus, it seems unlikely that the viral genome plays any significant role in the phenomenon. Published data indicate that other viruses also show some kind of synergism with LPS, although by different cellular mechanisms. T cells and their IFN-gamma production--crucial in the synergism of influenza viruses and LPS--were dispensable in our experiments. We suggest that the phenomenon is probably a general one: an overlap between different molecular mechanisms detecting bacterial and viral pathogens and inducing mediators of nonspecific cell-mediated host defense. The synergism of viruses and LPS (bacteria) could be a concern in medical practice as well as in gene therapy experiments with high doses of recombinant adenoviruses.

A novel synthetic, nonpsychoactive cannabinoid acid (HU‐320) with antiinflammatory properties in murine collagen‐induced arthritis

To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University-320 (HU-320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU-320 on macrophages and lymphocytes. DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU-320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU-320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU-320 on production of tumor necrosis factor alpha (TNF alpha) and reactive oxygen intermediates (ROIs). The effect of HU-320 administration on lipopolysaccharide-induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU-320 psychoactivity was performed using established laboratory tests on Sabra mice. Systemic daily administration of 1 and 2 mg/kg HU-320 ameliorated established CII-induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII-specific and polyclonal responses of murine and human lymphocytes were down-modulated. HU-320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU-320 administration yielded no adverse psychotropic effects in mice. Our studies show that the novel synthetic cannabinoid acid HU-320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.

Via β-adrenoceptors, stimulation of extrasplenic sympathetic nerve fibers inhibits lipopolysaccharide-induced TNF secretion in perfused rat spleen

Using a spleen slice microsuperfusion technique in mice, we have previously characterized the role of norepinephrine (NE) and other neurotransmitters for sympathetic modulation of IL-6 and TNF secretion of splenic macrophages. Since experiments in spleen slices do not reflect the situation of an entire perfused organ, we investigated sympathetic modulation of lipopolysaccharide (LPS)-induced secretion of IL-6 and TNF in perfusion experiments of rat spleen. In an organ bath, perfusion was performed in explanted whole spleens, and catecholamines and cytokines were measured by HPLC and ELISA, respectively. Release of NE depended on stimulation frequency (maximum at 10 Hz). Apart from NE, perfusates also contained significant amounts of dopamine and epinephrine. Furthermore, perfusate epinephrine levels correlated positively with perfusate NE levels ( R Rank=0.750, p<0.001) but not with plasma epinephrine concentrations. This indicates that epinephrine is a conversion product of sympathetically released NE. Early electrical stimulation of extrasplenic splenic nerves, 20 min after administration of LPS, significantly inhibited TNF secretion. This electrically induced effect was abrogated by simultaneous administration of propranolol (10 -6 M) but it was not influenced by administration of either an α1- or α2-adrenergic antagonist. Late electrical stimulation of splenic nerves, 2.5 h after administration of LPS, did not change TNF secretion. Interestingly, no influence of early or late sympathetic nerve fiber stimulation on IL-6 secretion was observed. In conclusion, this is the first perfusion study of the entire spleen that demonstrates that early electrical stimulation of sympathetic splenic nerve fibers directly inhibits LPS-induced TNF secretion. This study corroborates the idea that splenic sympathetic nerves are able to inhibit important activators of the innate immune system when stimulation happens very early or even prior to their induction by LPS.

Regulation of newborn and adult monocytes by PGE2: evidence of comparable differentiation status.

PGE2, which is produced in large amounts by monocytes, is considered to be an important autocrine factor in the regulation of the immune response. Altered sensitivity to PGE2 is known to reflect the differentiation status of monocytes/macrophages. Since newborn monocytes are thought to be less differentiated than adult peripheral blood monocytes, this study was designed to determine whether the diminished newborn monocyte function, relative to adult peripheral blood monocytes, might be the result of an altered autocrine responsiveness to PGE2. Phosphodiesterase activity as well as cAMP and TNF alpha levels in response to PGE2 were measured in newborn and adult monocytes, as an index of sensitivity to PGE2. Basal intracellular cAMP levels were reduced in cord monocytes compared with adult monocytes. Addition of increasing concentrations of PGE2 (10M(-10)-10M-6) caused similar fold increases in intracellular levels of cAMP for both populations. Lipopolysaccharide-induced TNF alpha production was significantly reduced in a similar fashion for both cord and adult monocytes at high (> or = 10(-7)M) PGE2 concentration. Phosphodiesterase activity was comparable in lysates from both populations. This study shows that PGE2 does not differentially influence newborn and adult monocyte responses and therefore that the diminished functional abilities of newborn monocytes is not due to altered autocrine responsiveness.