Bile Acid G Protein-Coupled Membrane Receptor TGR5 Modulates Aquaporin 2-Mediated Water Homeostasis.

BACKGROUND: The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms. METHODS: We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout (Tgr5-/-) mice. RESULTS: Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5-/- mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated Tgr5-/- mice, LCA treatment failed to prevent reduction of AQP2 expression. CONCLUSIONS: TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney.

Constitutive expression of the anti-apoptotic Bcl-2 family member A1 in murine endothelial cells leads to transplant tolerance.

Anti-apoptotic genes, including those of the Bcl-2 family, have been shown to have dual functionality inasmuch as they inhibit cell death but also regulate inflammation. Several anti-apoptotic molecules have been associated with endothelial cell (EC) survival following transplantation; however, their exact role has yet to be elucidated in respect to controlling inflammation. In this study we created mice expressing murine A1 (Bfl-1), a Bcl-2 family member, under the control of the human intercellular adhesion molecule 2 (ICAM-2) promoter. Constitutive expression of A1 in murine vascular ECs conferred protection from cell death induced by the proinflammatory cytokine tumour necrosis factor (TNF)-α. Importantly, in a mouse model of heart allograft transplantation, expression of A1 in vascular endothelium increased survival in the absence of CD8+ T cells. Better graft outcome in mice receiving an A1 transgenic heart correlated with a reduced immune infiltration, which may be related to increased EC survival and reduced expression of adhesion molecules on ECs. In conclusion, constitutive expression of the anti-apoptotic molecule Bfl1 (A1) in murine vascular ECs leads to prolonged allograft survival due to modifying inflammation.

Apremilast is a selective PDE4 inhibitor with regulatory effects on innate immunity.

Apremilast, an oral small molecule inhibitor of phosphodiesterase 4 (PDE4), is in development for chronic inflammatory disorders, and has shown efficacy in psoriasis, psoriatic arthropathies, and Behçet's syndrome. In March 2014, the US Food and Drug Administration approved apremilast for the treatment of adult patients with active psoriatic arthritis. The properties of apremilast were evaluated to determine its specificity, effects on intracellular signaling, gene and protein expression, and in vivo pharmacology using models of innate and adaptive immunity. Apremilast inhibited PDE4 isoforms from all four sub-families (A1A, B1, B2, C1, and D2), with IC50 values in the range of 10 to 100 nM. Apremilast did not significantly inhibit other PDEs, kinases, enzymes, or receptors. While both apremilast and thalidomide share a phthalimide ring structure, apremilast lacks the glutarimide ring and thus fails to bind to cereblon, the target of thalidomide action. In monocytes and T cells, apremilast elevated intracellular cAMP and induced phosphorylation of the protein kinase A substrates CREB and activating transcription factor-1 while inhibiting NF-κB transcriptional activity, resulting in both up- and down-regulation of several genes induced via TLR4. Apremilast reduced interferon-α production by plasmacytoid dendritic cells and inhibited T-cell cytokine production, but had little effect on B-cell immunoglobulin secretion. In a transgenic T-cell and B-cell transfer murine model, apremilast (5mg/kg/day p.o.) did not affect clonal expansion of either T or B cells and had little or no effect on their expression of activation markers. The effect of apremilast on innate immunity was tested in the ferret lung neutrophilia model, which allows monitoring of the known PDE4 inhibitor gastrointestinal side effects (nausea and vomiting). Apremilast significantly inhibited lung neutrophilia at 1mg/kg, but did not induce significant emetic reflexes at doses <30 mg/kg. Overall, the pharmacological effects of apremilast are consistent with those of a targeted PDE4 inhibitor, with selective effects on innate immune responses and a wide therapeutic index compared to its gastrointestinal side effects.

No association of vitamin D intake or 25-hydroxyvitamin D levels in childhood with risk of islet autoimmunity and type 1 diabetes: the Diabetes Autoimmunity Study in the Young (DAISY).

AIMS/HYPOTHESIS: The aim of the study was to investigate the association between vitamin D intake and status and the risk of islet autoimmunity (IA) and subsequent type 1 diabetes in children at increased risk of type 1 diabetes. METHODS: The Diabetes Autoimmunity Study in the Young (DAISY) in Denver, CO, USA, has been following children at increased risk of diabetes since 1993. As of February 2011, 198 children developed IA during follow-up of 2,644 DAISY children. Vitamin D intake and plasma 25-hydroxyvitamin D [25(OH)D] were measured longitudinally. Proportional hazards regression analyses of time to IA, or type 1 diabetes in IA-positive children, were conducted, with vitamin D intake and 25(OH)D as time-varying covariates. HRs were calculated for a standard deviation difference in exposure, with adjustment for confounders. RESULTS: Intake of vitamin D was not associated with the risk of IA (adjusted HR 1.13; 95% CI 0.95, 1.35; p = 0.18) nor progression to diabetes in IA-positive children (adjusted HR 1.30; 95% CI 0.91, 1.86; p = 0.15). Moreover, 25(OH)D level was not associated with the risk of IA (adjusted HR 1.12; 95% CI 0.88, 1.43; p = 0.36), nor progression to diabetes in IA-positive children (adjusted HR 0.91; 95% CI 0.68, 1.22; p = 0.54). In the 128 children in whom we measured 25(OH)D at 9 months of age, 25(OH)D was not associated with risk of IA (n = 30 IA-positive children) (adjusted HR 1.02; 95% CI 0.96, 1.07; p = 0.58). CONCLUSIONS/INTERPRETATION: Neither vitamin D intake nor 25(OH)D levels throughout childhood were associated with the risk of IA or progression to type 1 diabetes in our population.

The E2A-HLF oncogenic fusion protein acts through Lmo2 and Bcl-2 to immortalize hematopoietic progenitors.

The oncogenic fusion protein E2A-HLF is a chimeric transcription factor that arises from the t(17;19) translocation in childhood B-cell acute lymphoblastic leukemias (B-precursor ALL) and is associated with very poor outcome. We show that retroviral-mediated expression of E2A-HLF alone is sufficient to immortalize primary lymphoid progenitors. We identify Lmo2 and Bcl-2 as direct target genes downstream of E2A-HLF. We use real-time PCR analysis to show that LMO2 and BCL-2 expression is preferentially upregulated both in biopsy material from t(17;19) B-precursor ALL patients and lymphoid cell lines derived from t(17;19) leukemias. Co-expression of Lmo2 and Bcl-2 was sufficient to immortalize lymphoid progenitor cells resulting in a similar phenotype to that induced by E2A-HLF alone. Both shRNA-mediated knockdown of Lmo2 expression and pharmacological inhibition of BCL-2 function in E2A-HLF immortalized cells severely compromised their viability. These data suggest that both Lmo2 and Bcl-2 are required for the action of E2A-HLF in leukemogenesis.

Tnfaip8 is an essential gene for the regulation of glucocorticoid-mediated apoptosis of thymocytes.

Glucocorticoids have significant immunoregulatory actions on thymocytes and T cells and act by binding and activating cytosolic glucocorticoid receptors, which translocate to the nucleus and control gene expression through binding to specific response elements in target genes. Glucocorticoids promote cell death by activating an apoptotic program that requires transcriptional regulation. We set out to identify genes that are crucial to the process of glucocorticoid-mediated thymocyte apoptosis. Freshly isolated murine primary thymocytes were treated with dexamethasone, mRNA isolated and used to screen DNA microarrays. A set of candidate genes with upregulated expression was identified and selected members assayed in reconstituted fetal thymic organ culture (FTOC). Fetal liver-derived hematopoietic progenitor cells (HPCs) were infected with retroviruses expressing individual genes then used to repopulate depleted fetal thymic lobes. Reconstituted FTOCs expressing the gene Tnfaip8 were treated with dexamethasone and shown to be greatly sensitized to dexamethasone. Retrovirus-mediated RNA interference was applied to knock down Tnfaip8 expression in HPCs and these were used to reconstitute FTOCs. We observed that downregulating the expression of Tnfaip8 alone was sufficient to effectively protect thymocytes against glucocorticoid-induced apoptosis. We propose that Tnfaip8 is crucial in regulating glucocorticoid-mediated apoptosis of thymocytes.

Identification of a novel cyclin required for the intrinsic apoptosis pathway in lymphoid cells.

We have identified an early step common to pathways activated by different forms of intrinsic apoptosis stimuli. It requires de novo synthesis of a novel cyclin, cyclin O, that forms active complexes primarily with Cdk2 upon apoptosis induction in lymphoid cells. Cyclin O expression precedes glucocorticoid and gamma-radiation-induced apoptosis in vivo in mouse thymus and spleen, and its overexpression induces caspase-dependent apoptosis in cultured cells. Knocking down the endogenous expression of cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis due to a failure in the activation of apical caspases while leaving CD95 death receptor-mediated apoptosis intact. Our data demonstrate that apoptosis induction in lymphoid cells is one of the physiological roles of cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle, the DNA damage checkpoints or transcriptional response to glucocorticoids.

Improving efficiency and access to mental health care: combining integrated care and advanced access.

OBJECTIVE: To provide an example of implementation of a new program that enhances access to mental health care in primary care. METHOD: A general and specialized mental health service was redesigned to introduce open access to comprehensive mental health care in a primary care clinic. Key variables measured before and after implementation of the clinic included numbers of completed referrals, waiting time for appointments and clinic productivity. Workload and pre/post-implementation waiting time data were gathered through a computerized electronic monitoring system. RESULTS: Waiting time for new appointments was shortened from a mean of 33 days to 19 min. Clinician productivity and evaluations of new referrals more than doubled. These improvements have been sustained for 4 years. CONCLUSION: Moving mental health services into primary care, initiating open access and increasing use of technological aids led to dramatic improvements in access to mental health care and efficient use of resources. Implementation and sustainability of the program were enhanced by using a quality improvement approach.

Common polymorphisms of the PAI1 gene do not play a major role in the development of diabetic nephropathy in Type 1 diabetes.

AIM: Plasminogen activator inhibitor 1 (PAI1) plays a key role in the regulation of extracellular matrix (ECM) degradation. ThePAI1 gene is therefore an excellent candidate gene for diabetic nephropathy. The aim of this study was to employ gene resequencing to identify common DNA polymorphisms in thePAI1gene, and subsequently assess haplotype tagged single nucleotide polymorphisms(htSNPs) using a case control design. METHODS All nine exons, exon-intron boundaries, introns 1, 4 and 7 and approximately 3 kb upstream and 5 kb downstream of thePAI1 gene were screened for DNA polymorphisms in 15 case and 15 control subjects using WAVE denaturing high-performance liquid chromatography technology and confirmed by DNA sequencing. Polymorphisms were genotyped in 86 healthy individuals using direct sequencing and haplotype tagged single nucleotide polymorphisms (htSNPs) identified. Genotyping of the htSNPs was performed in 583 Type 1 diabetic patients (222 with nephropathy, 361 without nephropathy)using Pyrosequencing. RESULTS: Twenty-one polymorphisms with a minor allele frequency (MAF)>1%were identified; 14 had a MAF> or =10%. Five htSNPs [c.-1968_69insG, c.43 G-->A (Ala15Thr), c.1092-105 A-->G, c.*1737 G-->A, c.*3711 C-->T] were identified. Haplotype frequencies were similar in case and control groups (likelihood ratio chi2 test,P=0.66). CONCLUSION: It is unlikely that common polymorphisms of thePAI1 gene strongly influence susceptibility to diabetic nephropathy in the White Type 1 diabetic population.

TEL-AML1 preleukemic activity requires the DNA binding domain of AML1 and the dimerization and corepressor binding domains of TEL.

The t(12;21)(p13;q22) translocation generates the TEL-AML1 (TEL, translocation-Ets-leukemia; AML1, acute myeloid leukemia-1) (ETV6-RUNX1) fusion product and is the most common chromosomal abnormality in pediatric leukemia. Our previous studies using a murine fetal liver transplantation model demonstrated that TEL-AML1 promotes the self-renewal of B-cell precursors in vitro and enhances the expansion of hematopoietic stem cells (HSCs) in vivo. This is consistent with the hypothesis that TEL-AML1 induces expansion of a preleukemic clone. Several studies have described domains within TEL-AML1 involved in the transcriptional regulation of specific target genes. However, it is unclear which of these domains is important for the activity of TEL-AML1 in preleukemic hematopoiesis. In order to examine this, we have generated a panel of deletion mutants and expressed them in HSCs. These experiments demonstrate that TEL-AML1 requires multiple domains from both TEL and AML1 to alter hematopoiesis. Furthermore, mutation of a single amino-acid residue within the runt homology domain of AML1, required for DNA binding, was sufficient to abrogate TEL-AML1 activity. These data suggest that TEL-AML1 acts as an aberrant transcription factor to perturb multiple pathways during hematopoiesis.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Connective tissue growth factor [CTGF]/CCN2 stimulates mesangial cell migration through integrated dissolution of focal adhesion complexes and activation of cell polarization.

Connective tissue growth factor [CTGF]/CCN2 is a prototypic member of the CCN family of regulatory proteins. CTGF expression is up-regulated in a number of fibrotic diseases, including diabetic nephropathy, where it is believed to act as a downstream mediator of TGF-beta function; however, the exact mechanisms whereby CTGF mediates its effects remain unclear. Here, we describe the role of CTGF in cell migration and actin disassembly in human mesangial cells, a primary target in the development of renal glomerulosclerosis. The addition of CTGF to primary mesangial cells induced cell migration and cytoskeletal rearrangement but had no effect on cell proliferation. Cytoskeletal rearrangement was associated with a loss of focal adhesions, involving tyrosine dephosphorylation of focal adhesion kinase and paxillin, increased activity of the protein tyrosine phosphatase SHP-2, with a concomitant decrease in RhoA and Rac1 activity. Conversely, Cdc42 activity was increased by CTGF. These functional responses were associated with the phosphorylation and translocation of protein kinase C-zeta to the leading edge of migrating cells. Inhibition of CTGF-induced protein kinase C-zeta activity with a myristolated PKC-zeta inhibitor prevented cell migration. Moreover, transient transfection of human mesangial cells with a PKC-zeta kinase inactive mutant (dominant negative) expression vector also led to a decrease in CTGF-induced migration compared with wild-type. Furthermore, CTGF stimulated phosphorylation and activation of GSK-3beta. These data highlight for the first time an integrated mechanism whereby CTGF regulates cell migration through facilitative actin cytoskeleton disassembly, which is mediated by dephosphorylation of focal adhesion kinase and paxillin, loss of RhoA activity, activation of Cdc42, and phosphorylation of PKC-zeta and GSK-3beta. These changes indicate that the initial stages of CTGF mediated mesangial cell migration are similar to those involved in the process of cell polarization. These findings begin to shed mechanistic light on the renal diabetic milieu, where increased CTGF expression in the glomerulus contributes to cellular dysfunction.

Effects of SP500263, a novel selective estrogen receptor modulator, on bone, uterus, and serum cholesterol in the ovariectomized rat.

We describe here the activity of a novel selective estrogen receptor modulator, SP500263. When given to adult ovariectomized (OVX) rats for 28 days at doses of 0.3, 1, or 3 mg/kg/day, we found that SP500263 partially protected against OVX-induced loss of bone mineral content in the distal ends of femurs and in the whole bone. SP500263 also antagonized the OVX-induced increase in body weight. However, unlike 17beta-estradiol, SP500263 at efficacious doses did not prevent the OVX-induced loss in uterine wet weight. A small but significant effect on uterine wet weight was noted with raloxifene dosed at 1 mg/kg. As expected, SP500263 but not raloxifene acted as an estrogen antagonist on the uterus in adult rats when administered for 7 days at 30 mg/kg/day. Finally, SP500263 had no statistically significant effects on total serum cholesterol and serum triglycerides in OVX rats treated for 28 days. Raloxifene had no significant effects on body weight, bone mineral content, and serum cholesterol or triglycerides in the OVX-rat model. In summary, SP500263 is a new orally active SERM that acts in rats as an estrogen agonist on bone without causing uterine stimulatory effects.

Matua bromegrass hay for mares in gestation and lactation.

Matua bromegrass hay (Bromus willdenowii Kunth) is a high quality forage, but its value for mares during gestation and lactation is not well known. Intake, rate of passage, performance, and reproduction by gestating and lactating Quarter Horse mares fed the hay was investigated. In this experiment, 12, 2- to 12-yr-old gravid mares (mean BW = 553 kg, SD = 36) were fed Matua hay (CP = 11.5%) or alfalfa hay (Medicago sativa L.) (CP = 15.4%) for variable days prepartum (mean 59.9 d; SD = 23.5) and for 70 d postpartum. Matua and alfalfa hay were fed as the roughage portion of the diet with a grain supplement. Mares, blocked by age, expected date of foaling, and BW, were assigned randomly within blocks to treatments (six mares per treatment). Forage type did not affect intake, gestation length, birth weight, number of foals, foal weight gain, day of first postpartum ovulation, cycles per conception, or pregnancy rate at 70 d. On d 1, milk from mares fed alfalfa hay contained less (P < 0.03) CP than milk from mares fed Matua hay. Milk CP decreased (P < 0.01) in all mares over time. In a separate experiment, voluntary intake and rate of passage of Matua (CP = 15.5%), alfalfa (CP = 24.9%), and Timothy (Phleum pratense L.) (CP = 4.1%) hays were determined in nine 2-yr-old pregnant mares (mean BW = 447 kg; SD = 21). Diets were 100% forage. Timothy hay did not meet CP requirements for mares. Voluntary intake of alfalfa hay was higher (P < 0.01) than Matua hay. Intake of Timothy hay was lower (P < 0.01) than the mean of alfalfa and Matua hay. Rate of passage offorage was measured by passage of Cr-mordanted fiber. Passage rate and retention time did not differ between Matua and alfalfa hay; however, the retention times of Matua and alfalfa hays were shorter (P < 0.01) than for Timothy hay. Our results indicate that Matua hay is a forage that can be used safely for mares during gestation and early lactation and for their young foals.

Posture-related tachycardia in older patients with hyponatremia.

Hyponatremia (HN) is the commonest electrolyte abnormality in elderly patients. Its etiology in this setting is poorly understood. In this study, the authors aim to compare the hemodynamic and hormonal responses of a group of older patients with a predisposition to HN with a group of age-matched controls. We assessed hemodynamic and hormonal responses to postural challenge in 15 patients over age 65 with serum sodium concentrations of less than 130 mM (mean 128.7 mM) and 15 age-matched controls with normal sodium concentrations. Patients remained recumbent for 1 h and stood for the second. Blood was drawn at baseline and at 15 min intervals. Blood pressure (BP) and pulse rates (PR) were monitored electronically. Plasma arginine vasopressin (AVP), atrial natriuretic peptide (ANP), renin and aldosterone were determined periodically during the study period. No difference in BP between groups was noted. PR increased significantly in the HN group only within 3 min of standing (from 71 +/- 4 to 86 +/- 5, P<0.01) and remained significantly higher than controls until 90 min (87 +/- 5 vs. 69 +/- 4, P<0.01). While plasma AVP levels increased significantly following 30 min standing and remained elevated for both HN and control groups, it did not differ significantly between the two. Baseline plasma ANP levels were significantly higher in HN patients compared with controls and remained significantly higher (P<0.05) throughout the study. There was no significant difference in plasma renin or aldosterone levels between groups during the study period. We have demonstrated differing autonomic and hormonal responses to orthostatic challenge between HN patients and age-matched controls. Water retention due to the syndrome of inappropriate anti-diuretic hormone secretion (with reset osmostat) may lead to raised ANP levels in this older cohort of patients. Further physiological studies are required to clarify the precise mechanism of these varying responses.

Priming prostate carcinoma cells for increased apoptosis is associated with up-regulation of the caspases.

BACKGROUND: The potential to prime prostatic carcinoma cell lines for apoptosis represents an exciting strategy for the treatment of patients with this disease. The ability and the underlying molecular mechanisms involved in sensitizing both androgen-sensitive and androgen-insensitive cell types to a range of apoptotic-inducing agents are investigated by the authors. METHODS: Primary and secondary cell lines were pretreated with diethyl-maleate (DEM) prior to the induction of apoptosis by Fas antibody (1 microg/mL), cycloheximide (1 microg/mL), etoposide (62.5 microM), and radiation (5 grays). It was demonstrated previously that DEM (50 microM) increases the sensitivity to apoptosis induced by these agents. The effects of DEM on both protein and RNA expression was determined by Western blot analysis and a ribonuclease protection assay, respectively. The effects of DEM on intracellular glutathione (GSH) levels and its intracellular distribution also were assessed. RESULTS: DEM did not affect the expression of the caspases at the transcriptional level but was associated with increased procaspase-3 and caspase-8 protein levels. DEM preincubation restored sensitivity to Fas antibody and radiation-induced apoptosis in cells from the LNCaP-bcl-2 transfectant cell line that, normally, are resistant to these apoptotic stimuli. It is that DEM chemically depletes intracellular thiol levels. Although no depletion in total intracellular thiol GSH was observed at these concentrations of DEM, trafficking of GSH from the nucleus to the cytosol was demonstrated. CONCLUSIONS: Identification of the caspases as a potential target for chemical manipulation may serve as an effective, adjuvant-based approach in the treatment of patients with prostate carcinoma and, in particular, for immunotherapy and radiation-based strategies that rely on the activation of these death-effector proteases.

Lipoxin A(4) and aspirin-triggered 15-epi-lipoxin A(4) antagonize TNF-alpha-stimulated neutrophil-enterocyte interactions in vitro and attenuate TNF-alpha-induced chemokine release and colonocyte apoptosis in human intestinal mucosa ex vivo.

Lipoxins (LXs) are lipoxygenase-derived eicosanoids and putative endogenous braking signals for inflammation in the gastrointestinal tract and other organs. Aspirin triggers the production of 15-epimers during cell-cell interaction in a cytokine-primed milieu, and aspirin-triggered 15-epi-5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (15-epi-LXA(4)) may contribute to the bioactivity profile of this prototype nonsteroidal anti-inflammatory drug in vivo. We determined the effect of LXA(4), 15-(R/S)-methyl-11,12-dehydro-LXA(4) methyl ester (15-(R/S)-methyl-LXA(4)), and stable analogs of LXA(4) on TNF-alpha-stimulated neutrophil-enterocyte interaction in vitro and TNF-alpha-stimulated chemokine release, changes in mucosal architecture, and enterocyte apoptosis in cytokine-activated intact human colonic mucosa ex vivo. LXA(4), 15-(R/S)-epi-LXA(4), and 16-phenoxy-11,12-dehydro-17,18,19,20-tetranor-LXA(4) methyl ester (16-phenoxy-LXA(4)) inhibited TNF-alpha-stimulated neutrophil adherence to epithelial monolayers at nanomolar concentrations. In parallel experiments involving human colonic mucosa ex vivo, LXA(4)potently attenuated TNF-alpha-stimulated release of the C-X-C chemokine IL-8, and the C-C chemokines monocyte-chemoattractant protein-1 (MCP-1) and RANTES. Exposure of strips of normal human colonic mucosa to TNF-alpha induced disruption of mucosa architecture and enhanced colonocyte apoptosis via a caspase-3-independent mechanism. Prior exposure of the mucosa strips to 15-(R/S)-methyl-LXA(4) attenuated TNF-alpha-stimulated colonocyte apoptosis and protected the mucosa against TNF-alpha-induced mucosal damage. In aggregate, our data demonstrate that lipoxins and aspirin-triggered 15-epi-LXA(4) are potent antagonists of TNF-alpha-mediated neutrophil-enterocyte interactions in vitro, attenuate TNF-alpha-triggered chemokine release and colonocyte apoptosis, and are protective against TNF-alpha-induced morphological disruption in human colonic strips ex vivo. Our observations further expand the anti-inflammatory profile of these lipoxygenase-derived eicosanoids and suggest new therapeutic approaches for the treatment of inflammatory bowel disease.

Lipoxins: revelations on resolution.

Lipoxins (LXs) are endogenously produced eicosanoids typically generated during cell-cell interactions. In this article, the compelling evidence from in vitro and in vivo model systems that LXs actively promote the resolution of inflammation is reviewed. Of particular interest are indications that stable synthetic analogues of LXs and aspirin-triggered 15-epi-LXs can mimic many of the desirable anti-inflammatory, "pro-resolution" actions of native LXs. Given the enhanced stability and efficacy of these compounds a role as novel anti-inflammatory therapeutics is proposed.