Necrosis Factor Ligand Superfamily Member Research Articles

Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12−/−ApoE−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12−/−ApoE−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12+/+ApoE−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12−/−ApoE−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12+/+ApoE−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12−/−ApoE−/− or anti-TWEAK treatment in TNFSF12+/+ApoE−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.

Genetic risk and protective factors of TNFSF15 gene variants detected using single nucleotide polymorphisms in Hungarians with psoriasis and psoriatic arthritis

The aim of this study was to examine the role of single nucleotide polymorphisms (SNPs) and haplotypes of the tumor necrosis factor ligand superfamily member 15 (TNFSF15) gene in Hungarians with psoriasis and psoriatic arthritis. A case-control study was performed, and five TNFSF15 SNPs (rs3810936, rs6478108, rs6478109, rs7848647, rs7869487) were genotyped in 319 patients with psoriasis, 105 of whom also have psoriatic arthritis, and in 200 healthy individuals. Three haplotypes (A, B, C) based on these five SNPs were also analyzed. Our findings suggest that the rs6478109 SNP may be a genetic risk factor in psoriasis (p=0.0046), while haplotype C may be protective (p=0.0250). These results suggest that certain variants of the TNFSF15 gene contribute to the pathogenesis of the immune-mediated, multifactorial skin disease psoriasis, and that this difference is more readily apparent when groups of patients with and without psoriatic arthritis are examined separately.

Immune effects of dietary anethole on Eimeria acervulina infection

The effects of anethole on in vitro and in vivo parameters of chicken immunity during experimental avian coccidiosis were evaluated. Anethole reduced the viability of invasive Eimeria acervulina sporozoites after 2 or 4 h of treatment in vitro by 45 and 42%, respectively, and stimulated 6.0-fold greater chicken spleen cell proliferation compared with controls. Broiler chickens continuously fed from hatch with an anethole-supplemented diet and orally challenged with live E. acervulina oocysts showed enhanced BW gain, decreased fecal oocyst excretion, and greater E. acervulina profilin antibody responses compared with infected chickens given an unsupplemented standard diet. The levels of transcripts encoding the immune mediators IL6, IL8, IL10, and tumor necrosis factor ligand superfamily member 15 (TNFSF15) in intestinal lymphocytes were increased in E. acervulina-infected chickens fed the anethole-containing diet compared with untreated controls. Global gene expression analysis by microarray hybridization identified 1,810 transcripts (677 upregulated, 1,133 downregulated) whose levels were significantly altered in intestinal lymphocytes of anethole-fed birds compared with unsupplemented controls. From this transcriptome, 576 corresponding genes were identified. The most significant biological function associated with these genes was "Inflammatory Response" in the "Disease and Disorders" category. This new information documents the immunologic and genomic changes that occur in chickens following anethole dietary supplementation that may be relevant to host protective immune response to avian coccidiosis.

MicroRNA regulate immune pathways in T-cells in multiple sclerosis (MS)

BackgroundMicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls.MethodsHeparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells. MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsWe identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls. By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS. The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA.ConclusionThese findings indicate that microRNA may be important regulatory molecules in T-cells in MS.

Association of TNFSF4 polymorphisms with systemic lupus erythematosus: a meta-analysis

Introduction To more precisely estimate the association between the tumor necrosis factor ligand superfamily member 4 (TNFSF4) gene polymorphisms and systemic lupus erythematosus (SLE) risk, we surveyed studies on the association of the TNFSF4 rs2205960, rs1234315, rs844644, and rs844648 polymorphisms with SLE.Methods A literature-based search was conducted to identify all relevant studies. A total of eight independent studies were identified and subsequently reviewed in the meta-analysis.Results The meta-analysis showed an association between the TNFSF4 rs2205960 polymorphism and SLE in all subjects [ odds ratio (OR) 1.327, 95 % confidence interval (CI) 1.227–1.436, P < 0.001]. In a subgroup analysis by ethnicity, a significantly increased risk for SLE was associated with TNFSF4 rs2205960 T allele among patients of European (OR 1.254, 95 % CI 1.185–1.328, P < 0.001) and Asian ethnicity (OR 1.425, 95 % CI 1.352–1.501, P < 0.001). The meta-analysis of the rs1234315 polymorphism revealed no association between SLE and the rs1234315 T allele in all subjects (OR 1.167, 95 % CI 0.874–1.558, P = 0.296), but the results of the subgroup analysis revealed significant association in subjects of Asian ethnicity (OR 1.386, 95 % CI 1.318–1.458, P < 0.001). No association was found between the rs844644 and rs844648 polymorphisms and SLE.Conclusion The results of our meta-analysis suggest that the TNFSF4 rs2205960 polymorphism may confer susceptibility to SLE in different populations and that the TNFSF4 rs1234315 polymorphism is associated with susceptibility to SLE in Asians.

ImmunoChip Study Implicates Antigen Presentation to T Cells in Narcolepsy

Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin) deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.

Do γδ T cells predict osteonecrosis of the jaw?

A large body of evidence has established that T cells and cytokine mediators originating from the immune system potently impact bone turnover in both physiologic and pathologic conditions. Indeed, osteoclasts derive from precursors that circulate within the immune compartment; however, new revelations continue to emerge attesting to the depth of integration of the skeletal and immune systems, and the consequences of the immuno-skeletal interface for bone metabolism. Activated T cells have long been recognized to promote osteoclast formation as a consequence of their capacity to secrete osteoclastogenic cytokines including receptor activator of NF-kB ligand (RANKL)(1–5); the RANKL-independent osteoclastogenic cytokines, secreted osteoclastogenic factor of activated T cells (SOFAT)(6) and tumor necrosis factor (TNF) ligand superfamily member 14 (TNSF14, or LIGHT),(7) and the inflammatory mediator TNFα.(8) TNFα stimulates RANKL production in osteoblastic cells(9) and potently synergizes with RANKL to intensify osteoclastogenesis(8,10,11) and resorption.(12) Indeed, RANKL and/or TNFα, derived in part from activated T cells, have been implicated in bone loss and joint destruction in animal models of rheumatoid arthritis,(13) alveolar bone loss in periodontal infection,(14,15) and in humans and animal models of the archetypal osteoporotic bone disease, postmenopausal osteoporosis.(5,16,17) Interestingly, although lymphocytes are protagonists of bone loss in inflammatory states, under physiologic conditions both human(18) and rodent B cells(19,20) secrete the RANKL decoy receptor osteoprotegerin (OPG), an activity potentiated by T cell costimulation.(19) T cells are thus central to the regulation of basal bone turnover as well as protagonists of pathological bone loss.

Ampicillin-Improved Glucose Tolerance in Diet-Induced Obese C57BL/6NTac Mice Is Age Dependent

Ampicillin has been shown to improve glucose tolerance in mice. We hypothesized that this effect is present only if treatment is initiated prior to weaning and that it disappears when treatment is terminated. High-fat fed C57BL/6NTac mice were divided into groups that received Ampicillin at different ages or not at all. We found that both diet and Ampicillin significantly changed the gut microbiota composition in the animals. Furthermore, there was a significant improvement in glucose tolerance in Ampicillin-treated, five-week-old mice compared to nontreated mice in the control group. At study termination, expressions of mRNA coding for tumor necrosis factor, serum amyloid A, and lactase were upregulated, while the expression of tumor necrosis factor (ligand) superfamily member 15 was downregulated in the ileum of Ampicillin-treated mice. Higher dendritic cell percentages were found systemically in high-fat diet mice, and a lower tolerogenic dendritic cell percentage was found both in relation to high-fat diet and late Ampicillin treatment. The results support our hypothesis that a “window” exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning.

Association of TNFSF4 polymorphisms with systemic lupus erythematosus: a meta-analysis

To more precisely estimate the association between the tumor necrosis factor ligand superfamily member 4 (TNFSF4) gene polymorphisms and systemic lupus erythematosus (SLE) risk, we surveyed studies on the association of the TNFSF4 rs2205960, rs1234315, rs844644, and rs844648 polymorphisms with SLE. A literature-based search was conducted to identify all relevant studies. A total of eight independent studies were identified and subsequently reviewed in the meta-analysis. The meta-analysis showed an association between the TNFSF4 rs2205960 polymorphism and SLE in all subjects [ odds ratio (OR) 1.327, 95% confidence interval (CI) 1.227-1.436, P < 0.001]. In a subgroup analysis by ethnicity, a significantly increased risk for SLE was associated with TNFSF4 rs2205960 T allele among patients of European (OR 1.254, 95% CI 1.185-1.328, P < 0.001) and Asian ethnicity (OR 1.425, 95% CI 1.352-1.501, P < 0.001). The meta-analysis of the rs1234315 polymorphism revealed no association between SLE and the rs1234315 T allele in all subjects (OR 1.167, 95% CI 0.874-1.558, P = 0.296), but the results of the subgroup analysis revealed significant association in subjects of Asian ethnicity (OR 1.386, 95% CI 1.318-1.458, P < 0.001). No association was found between the rs844644 and rs844648 polymorphisms and SLE. The results of our meta-analysis suggest that the TNFSF4 rs2205960 polymorphism may confer susceptibility to SLE in different populations and that the TNFSF4 rs1234315 polymorphism is associated with susceptibility to SLE in Asians.

Altered expression of TNFSF4 and TRAF2 mRNAs in peripheral blood mononuclear cells in patients with systemic lupus erythematosus: association with atherosclerotic symptoms and lupus nephritis

This study compares the expression levels of tumor necrosis factor ligand superfamily member 4 (TNFSF4) and TNF-R-associated factor 2 (TRAF2) mRNAs in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) against healthy controls. The association of SLE disease activity index (SLEDAI) and clinical features of SLE with altered expression levels of TNFSF4 and TRAF2 mRNAs were also evaluated. We used real-time reverse transcription polymerase chain reaction to measure TNFSF4 and TRAF2 mRNAs expression levels in peripheral blood mononuclear cells of 57 SLE patients and 57 healthy controls. The expression level of TNFSF4 mRNA was significantly higher in SLE patients than in the control group. Overexpression of TNFSF4 was correlated with arthritis, atherosclerosis and lupus nephritis. TRAF2 mRNA was underexpressed in PBMCs of SLE patients, and its lower expression was associated with atherosclerosis and lupus nephritis. The altered expression levels of TNFSF4 and TRAF2 mRNAs was significantly correlated with SLEDAI. Our results suggest that changes in the expression levels of TNFSF4 and TRAF2 mRNAs may significantly correlate with the pathogenesis of SLE, the disease activity and different clinical features of lupus, particularly lupus nephritis, atherosclerosis and arthritis.

TRAF-6 Dependent Signaling Pathway Is Essential for TNF-Related Apoptosis-Inducing Ligand (TRAIL) Induces Osteoclast Differentiation

Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

TRAF-6 dependent signaling pathway is essential for TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation (54.21)

Abstract Human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (TNF) ligand superfamily members and their receptors. Recent evidence indicates that in addition to triggering apoptosis, the TNF-related apoptosis-inducing ligand (TRAIL) induces osteoclast differentiation. To understand TRAIL-mediated signal transduction mechanism in osteoclastogenesis, we demonstrated that TRAIL induces osteoclast differentiation via a Tumor necrosis factor receptor-associated factor 6 (TRAF-6)-dependent signaling pathway. TRAIL-induced osteoclast differentiation was significantly inhibited by treatment with TRAF-6 siRNA and TRAF6 decoy peptides in both human monocytes and murine RAW264.7 macrophage cell lines, as evaluated in terms of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and bone resorption activity. Moreover, TRAIL-induced osteoclast differentiation was also abolished in TRAF6 knockout bone marrow macrophages. In addition to induction of NFATc1, treatment of TRAIL also induced ubiquitination of TRAF6 in osteoclast differentiation. Thus, our data demonstrate that TRAIL induces osteoclastic differentiation via a TRAF-6 dependent signaling pathway. This study suggests TRAF6-dependent signaling may be a central pathway in osteoclast differentiation, and that TNF superfamily molecules other than RANKL may modify RANK signaling by interaction with TRAF6-associated signaling.

Characterisation and expression analysis of B-cell activating factor (BAFF) in spiny dogfish (Squalus acanthias): Cartilaginous fish BAFF has a unique extra exon that may impact receptor binding

B-cell activating factor (BAFF), also known as tumour necrosis factor (TNF) ligand superfamily member 13B, is an important immune regulator with critical roles in B-cell survival, proliferation, differentiation and immunoglobulin secretion. A BAFF gene has been cloned from spiny dogfish (Squalus acanthias) and its expression studied. The dogfish BAFF encodes for an anchored type-II transmembrane protein of 288 aa with a putative furin protease cleavage site and TNF family signature as seen in BAFFs from other species. The identity of dogfish BAFF has also been confirmed by conserved cysteine residues, and phylogenetic tree analysis. The dogfish BAFF gene has an extra exon not seen in teleost fish, birds and mammals that encodes for 29 aa and may impact on receptor binding. The dogfish BAFF is highly expressed in immune tissues, such as spleen, and is up-regulated by PWM in peripheral blood leucocytes, suggesting a potentially important role in the immune system.

Insights into the pathogenesis of systemic sclerosis based on the gene expression profile of progenitor‐derived endothelial cells

To determine the gene expression profile of endothelial cells derived from the endothelial progenitor cells (EPCs) of patients with systemic sclerosis (SSc). Microarray experiments were performed on Affymetrix GeneChip Human Exon 1.0 ST Arrays in unstimulated and hypoxia-stimulated EPC-derived cells from patients with SSc and control subjects. Followup of the raised hypotheses was performed ex vivo by immunohistochemical analysis of skin tissue. Signals from 92 probe sets and 188 probe sets were different in unstimulated and hypoxia-stimulated cells, respectively, from patients with SSc compared with controls. Within the largest groups of genes related to cell-cell interaction and vascular remodeling, down-regulation of tumor necrosis factor ligand superfamily member 10 (TNFSF10) and homeobox A9 (HOX-A9) was confirmed by real-time polymerase chain reaction and Western blots in EPC-derived cells and by immunohistochemistry in SSc skin tissue. Signals from 221 and 307 probe sets were different in unstimulated and hypoxia-stimulated cells, respectively, from patients with diffuse cutaneous SSc compared with patients with limited cutaneous SSc. Within the largest group of genes related to the inflammatory response, differential expression of TNFα-induced protein 3 and prostaglandin-endoperoxide synthase 2 was observed in EPC-derived cells and skin tissue from patients with SSc. Our data revealed important gene expression changes in EPC-derived endothelial cells from patients with SSc, characterized by a proadhesive, proinflammatory, and activated phenotype. Differential expression in lesional SSc skin tissue of new targets, such as TNF family members and HOX-A9, may contribute to the pathogenesis of SSc and deserves more in-depth exploration.

Tumor necrosis factor ligand superfamily member 13 (TNFSF13; APRIL)

Tumor necrosis factor ligand superfamily member 13 (TNFSF13; APRIL)

Tumor necrosis factor ligand superfamily member 14 (TNFSF14; LIGHT; CD258)

Tumor necrosis factor ligand superfamily member 14 (TNFSF14; LIGHT; CD258)

Denosumab: An update

Denosumab is a fully human monoclonal antibody that inhibits the formation, function and survival of osteoclasts, preventing the interaction of tumor necrosis factor ligand superfamily member 11 (receptor activator of nuclear factor kappa-B ligand, RANKL) with the tumor necrosis factor receptor superfamily member 11A (osteoclast differentiation factor receptor, ODFR, receptor activator of NF-KB, RANK). This results in a reduction in bone resorption and an increase in bone mineral density. In clinical studies, denosumab has been shown to decrease the risk for vertebral, hip and nonvertebral fractures in women with postmenopausal osteoporosis and the risk for new vertebral fractures in men with nonmetastatic prostate cancer receiving androgen deprivation therapy, with a rate of side effects similar to placebo. A number of clinical trials with denosumab are ongoing to demonstrate its value for other indications and to further characterize its effects on immunomodulation. Denosumab is a new alternative for the prevention and treatment of postmenopausal osteoporosis and a promising agent for the treatment of other bone diseases associated with bone loss.

Proteomic analysis of morphine rabbit myocardium with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

To analyze the morphine rabbit myocardium with matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six New Zealand white rabbits were randomly assigned to a control group (Group C) and a morphine group (Group M). Group C were pretreated with bolus injection of saline 1 mL/kg. Group M were pretreated with bolus injection of morphine 3 mg/kg. The myocardium tissue proteins of the rabbits 24 hours after the injection of morphine or saline preconditioned were extracted and separated by two dimensional gel electrophoresis(2-DE), and the images were analyzed and different proteins were found. Some of the different proteins were determined with MALDI-TOF-MS. There were 51 protein spots that displayed quantitative changes in expression (P < 0.05), 15 protein spots were chosen for MS analysis, and 8 proteins were preliminarily identified.They were aldose reductase, zinc finger protein 312, src related tyrosine kinase, carbonic anhydrase 12 precursor, electron transfer flavoprotein beta-subunit, glyceraldehyde-3-phosphate dehydrogenase, tumor necrosis factor ligand superfamily member 11 and transmembrane emp24 domain-containing protein. These proteins may be involved in the cardioprotection of morphine preconditioning.

Global gene expression profiles of ischemic preconditioning in deceased donor liver transplantation

The benefits of ischemic preconditioning (IPC) in reducing ischemia/reperfusion injury (IRI) remain indistinct in human liver transplantation (LT). To further understand mechanistic aspects of IPC, we performed microarray analyses as a nested substudy in a randomized trial of 10-minute IPC in 101 deceased donor LTs. Liver biopsies were performed after cold storage and at 90 minutes postreperfusion in 40 of 101 subjects. Global gene expression profiles in 6 biopsy pairs in IPC and work standard organ recovery groups at both time points were compared using the Affymetrix GeneChip Human Gene 1.0 ST array. Transcripts with >1.5-fold change and P < 0.05 were considered significant. IPC altered expression of 82 transcripts in antioxidant, immunological, lipid biosynthesis, cell development and growth, and other groups. Real-time polymerase chain reaction and immunoblotting validated our microarray data. IPC-induced overexpression of glutathione S-transferase mu transcripts (GSTM1, GSTM3, GSTM4, and GSTM5) was accompanied by increased protein expression and may contribute to a decrease in oxidative stress. However, the increased expression of fatty acid synthase may increase oxidative stress, and tumor necrosis factor ligand superfamily member 10 may promote apoptosis. These changes, in combination with decreased expression of heparin-binding epidermal growth factor-like growth factor and insulin-like growth factor binding protein-1, both of which inhibit apoptosis, may increase IRI. In our study of deceased donor LT, IPC induces changes in gene expression, some of which are potentially beneficial but some which are potentially injurious. Thus, our findings of changes in gene expression mirror the outcomes in our clinical trial.

A novel role for Glucocorticoid-Induced TNF Receptor Ligand (Gitrl) in early embryonic zebrafish development.

Tumour necrosis factor ligand and receptor superfamily (TNFSF and TNFRSF) members have diverse and well-studied functions in the immune system. Additional, non-immunological roles, such as in the morphogenesis of bone, tooth, hair and skin have also been described for some members. GITRL and its receptor GITR are well-described as co-regulators of the mammalian immune response. Here, we describe the identification and cloning of their zebrafish homologues and demonstrate a novel role for the ligand, but not the receptor, in early vertebrate development. The assignment of zebrafish Gitrl and Gitr was supported by homology and phylogenetic analysis. The ligand exhibited an oscillating pattern of mRNA expression during the first 36 hours post fertilization, during which time gitr mRNA was not detected, and morpholino oligonucleotide-mediated knock-down of gitrl, but not of gitr, resulted in disruption of early embryogenesis, most clearly revealed during gastrulation, which corresponded to the earliest peak in gitrl mRNA expression (5.25-10 hpf). We found Stat3 signalling to be altered in the gitrl-morphants, suggesting that one possible role for Gitrl during embryogenesis may be modulation of Jak/Stat signalling.