Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.

Association of a homozygous nonsense caveolin-1 mutation with Berardinelli-Seip congenital lipodystrophy

Context: Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue, resulting in severe dyslipidemia and insulin resistance. In most reported cases, BSCL is due to alterations in either seipin, of unknown function, or 1-acylglycerol-3- phosphate acyltransferase-beta (AGPAT2), which catalyzes the formation of phosphatidic acid. Objective: We sought to determine the genetic origin of the unexplained cases of BSCL. We thus sequenced CAV1, encoding caveolin-1, as a candidate gene involved in insulin signaling and lipid homeostasis. CAV1 is a key structural component of plasma membrane caveolae, and Cav1-deficient mice display progressive loss of adipose tissue and insulin resistance. Design: We undertook phenotyping studies and molecular screening of CAV1 in four patients with BSCL with no mutation in the genes encoding either seipin or AGPAT2. Results: A homozygous nonsense mutation (p.Glu38X) was identified in CAV1 in a patient with BSCL born from a consanguineous union. This mutation affects both the alpha-and beta-CAV1 isoforms and ablates CAV1 expression in skin fibroblasts. Detailed magnetic resonance imaging of the proband confirmed near total absence of both sc and visceral adipose tissue, with only vestigial amounts in the dorsal sc regions. In keeping with the lack of adipose tissue, the proband was also severely insulin resistant and dyslipidemic. In addition, the proband had mild hypocalcemia likely due to vitamin D resistance. Conclusions: These findings identify CAV1 as a new BSCL-related gene and support a critical role for caveolins in human adipocyte function.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A(1) receptors

Background and purpose: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. Experimental approach: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. Key results: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. Conclusions and implications: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

Inhibition of COX 1 and 2 prior to Renal Ischemia/Reperfusion Injury Decreases the Development of Fibrosis

Ischemia and reperfusion injury (IRI) contributes to the development of chronic interstitial fibrosis/tubular atrophy in renal allograft patients, Cyclooxygenase (COX) 1 and 2 actively participate in acute ischemic injury by activating endothelial cells and inducing oxidative stress. Furthermore, blockade of COX I and 2 has been associated with organ improvement after ischemic damage. The aim of this study was to evaluate the role of COX I and 2 in the development of fibrosis by performing a COX I and 2 blockade immediately before IRI We subjected C57BI/6 male mice to 60 min of unilateral renal pedicle occlusion, Prior to surgery mice were either treated with indomethacin (IMT) at days -1 and 0 or were untreated. Blood and kidney samples were collected 6 wks after IRI. Kidney samples were analyzed by real-time reverse transcription-poly me rase chain reaction for expression of transforming growth factor beta (TGF-beta), monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1 beta, IL-10, heme oxygenose 1 (HO-1), vimentin, connective-tissue growth factor (CTGF), collagen 1, and bone morphogenic protein 7 (BMP-7), To assess tissue fibrosis we performed morphometric analyses and Sirius red staining. We also performed immunohistochemical analysis of anti-actin smooth muscle, Renal function did not significantly differ between groups. Animals pretreated with IMT showed significantly less interstitial fibrosis than nontreated animals. Gene transcript analyses showed decreased expression of TGF-beta, MCP-1,TNF-alpha, IL-1-beta, vimentin, collagen 1, CTGF and IL-10 mRNA (all P < 0.05), Moreover, HO-I mRNA was increased in animals pretreated with IMT (P < 0.05) Conversely, IMT treatment decreased osteopontin expression and enhanced BMP-7 expression, although these levels did rot reach statistical significance when compared with control expression levels, I he blockade of COX 1 and 2 resulted in less tissue fibrosis, which was associated with a decrease in proinflammatory cytokines and enhancement of the protective cellular response.

9,10-Dihydroxy-1,8-cineole (1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octane-10,11-diol)

The title compound (3) has been synthesized and its presence sought in the urinary metabolites of the brushtail possum. © CSIRO 2001

Halogen-bonded tris (2,4-Bis(trichloromethyl)-1,3,5,triazapentadienato)-M(III) [M = Mn, Fe, Co] complexes and their catalytic activity in the peroxidative oxidation of 1-phenylethanol to acetophenone

One-pot template condensation of CCl3C=N with ammonia on a metal source [MnCl2 center dot 4H(2)O, FeCl3 center dot 6H(2)O or Co(CH3COO)(2)center dot 4H(2)O] in DMSO led to the formation of tris(2,4-bis(trichloromethyl)-1,3,5-triazapentadienato)-M(III) complexes, [M(NH=C(CCl3)NC(CCl3)=-NH}(3)]center dot n(CH3)(2)SO [M = Mn, n = 1 (1); M = Fe, n = 2 (2); M = Co, n = 2 (3)1, which were characterized using elemental analysis, and IR, ESI-MS and single-crystal X-ray analysis. The role of inter- and intramolecular non-covalent halogen and hydrogen bonds in the synthesis of 1-3 is discussed. It is shown that the crystal ionic radii of the metal ions [68.5 (Co) < 69 (Fe) < 72 (Mn), pm] are related to the corresponding Cl center dot center dot center dot Cl distances [3.178 (3) > 3.155 (2) > 3.133 (1) Al. Compounds 1-3 and the related di(triazapentadienato)-Cu(v) complex [Cu(NH=C(CCl3)NC(CCl3)=NH}2]center dot 2(CH3)(2)SO (4) act as catalyst precursors for the additive-free microwave (MW) assisted homogeneous oxidation of 1-phenylethanol with tert-butylhydroperoxide (TBHP), leading to the formation of acetophenone with yields up to 99% and TONs up to 5.0 x 10(3) after 1 h of low power (10 W) MW irradiation.

CD8 beta increases CD8 coreceptor function and participation in TCR-ligand binding.

To study the role of CD8 beta in T cell function, we derived a CD8 alpha/beta-(CD8-/-) T cell hybridoma of the H-2Kd-restricted N9 cytotoxic T lymphocyte clone specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260. This hybridoma was transfected either with CD8 alpha alone or together with CD8 beta. All three hybridomas released interleukin 2 upon incubation with L cells expressing Kd-peptide derivative complexes, though CD8 alpha/beta cells did so more efficiently than CD8 alpha/alpha and especially CD8-/- cells. More strikingly, only CD8 alpha/beta cells were able to recognize a weak agonist peptide derivative variant. This recognition was abolished by Fab' fragments of the anti-Kd alpha 3 monoclonal antibody SF1-1.1.1 or substitution of Kd D-227 with K, both conditions known to impair CD8 coreceptor function. T cell receptor (TCR) photoaffinity labeling indicated that TCR-ligand binding on CD8 alpha/beta cells was approximately 5- and 20-fold more avid than on CD8 alpha/a and CD8-/- cells, respectively. SF1-1.1.1 Fab' or Kd mutation D227K reduced the TCR photoaffinity labeling on CD8 alpha/beta cells to approximately the same low levels observed on CD8-/- cells. These results indicate that CD8 alpha/beta is a more efficient coreceptor than CD8alpha/alpha, because it more avidly strengthens TCR-ligand binding.

Phase I/IIa study of cilengitide and temozolomide with concomitant radiotherapy followed by cilengitide and temozolomide maintenance therapy in patients with newly diagnosed glioblastoma.

Purpose: Invasion and migration are key processes of glioblastoma and are tightly linked to tumor recurrence. Integrin inhibition using cilengitide has shown synergy with chemotherapy and radiotherapy in vitro and promising activity in recurrent glioblastoma. This multicenter, phase I/IIa study investigated the efficacy and safety of cilengitide in combination with standard chemoradiotherapy in newly diagnosed glioblastoma. Patients and Methods: Patients (age >= 18 to >= 70 years) were treated with cilengitide (500 mg) administered twice weekly intravenously in addition to standard radiotherapy with concomitant and adjuvant temozolomide. Treatment was continued until disease progression or for up to 35 weeks. The primary end point was progression-free survival (PFS) at 6 months. Results: Fifty-two patients ( median age, 57 years; 62% male) were included. Six- and 12-month PFS rates were 69% (95% CI, 54% to 80%) and 33% ( 95% CI, 21% to 46%). Median PFS was 8 months ( 95% CI, 6.0 to 10.7 months). Twelve- and 24-month overall survival ( OS) rates were 68% ( 95% CI, 53% to 79%) and 35% ( 95% CI, 22% to 48%). Median OS was 16.1 months ( 95% CI, 13.1 to 23.2 months). PFS and OS were longer in patients with tumors with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (13.4 and 23.2 months) versus those without MGMT promoter methylation (3.4 and 13.1 months). The combination of cilengitide with temozolomide and radiotherapy was well tolerated, with no additional toxicity. No pharmacokinetic interactions between temozolomide and cilengitide were identified. Conclusion: Compared with historical controls, the addition of concomitant and adjuvant cilengitide to standard chemoradiotherapy demonstrated promising activity in patients with glioblastoma with MGMT promoter methylation. J Clin Oncol 28:2712-2718. (C) 2010 by American Society of Clinical Oncology

Isolated integrin beta3 subunit cytoplasmic domains require membrane anchorage and the NPXY motif to recruit to adhesion complexes but do not discriminate between beta1- and beta3-positive complexes.

Integrin adhesion receptors consist of non-covalently linked alpha and beta subunits each of which contains a large extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Engaged integrins recruit to focal structures globally termed adhesion complexes. The cytoplasmic domain of the beta subunit is essential for this clustering. beta1 and beta3 integrins can recruit at distinct cellular locations (i.e. fibrillar adhesions vs focal adhesions, respectively) but it is not clear whether individual beta subunit cytoplasmic and transmembrane domains are by themselves sufficient to drive orthotopic targeting to the cognate adhesion complex. To address this question, we expressed full-length beta3 transmembrane anchored cytoplasmic domains and truncated beta3 cytoplasmic domains as GFP-fusion constructs and monitored their localization in endothelial cells. Membrane-anchored full-length beta3 cytoplasmic domain and a beta3 mutant lacking the NXXY motif recruited to adhesion complexes, while beta3 mutants lacking the NPXY and NXXY motifs or the transmembrane domain did not. Replacing the natural beta subunit transmembrane domain with an unrelated (i.e. HLA-A2 alpha chain) transmembrane domain significantly reduced recruitment to adhesion complexes. Transmembrane anchored beta3 and cytoplasmic domain constructs, however, recruited without discrimination to beta1- and beta3-rich adhesions complexes. These findings demonstrate that membrane anchorage and the NPXY (but not the NXXY) motif are necessary for beta3 cytoplasmic domain recruitment to adhesion complexes and that the natural transmembrane domain actively contributes to this recruitment. The beta3 transmembrane and cytoplasmic domains alone are insufficient for orthotopic recruitment to cognate adhesion complexes.

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients.

The paradoxical coexistence of spontaneous tumor antigen-specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1-specific CD8(+) T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1-specific CD8(+) T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1(+) NY-ESO-1-specific CD8(+) T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3(+)PD-1(+) NY-ESO-1-specific CD8(+) T cells are more dysfunctional than Tim-3(-)PD-1(+) and Tim-3(-)PD-1(-) NY-ESO-1-specific CD8(+) T cells, producing less IFN-γ, TNF, and IL-2. Tim-3-Tim-3L blockade enhanced cytokine production by NY-ESO-1-specific CD8(+) T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3-Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1-specific CD8(+) T cells upon prolonged antigen stimulation and acted in synergy with PD-1-PD-L1 blockade. Collectively, our findings support the use of Tim-3-Tim-3L blockade together with PD-1-PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

Characterization of a specific 20- to 25-kD interleukin-1 inhibitor from cultured human lung macrophages.

Alveolar macrophages have the ability to downregulate immune processes in vitro. We have recently suggested the presence of interleukin-1 (IL-1) inhibitors in the supernatants of human bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis or sarcoidosis. In the present study, we further analyze the cellular origin and the biologic properties of a 20- to 25-kD IL-1 inhibitor spontaneously produced by cultured human alveolar macrophages (AM). The inhibitor blocks IL-1-induced prostaglandin E2 production by human fibroblasts and the IL-1-related increase of phytohemagglutinin-induced murine thymocyte proliferation. After rigorous IL-1 alpha and IL-1 beta depletion, supernatants of lung macrophages specifically block the binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1 in a dose-dependent manner. These results indicate that AM from both normal donors and patients produce a specific IL-1 inhibitor that may be of importance in protecting the alveolar environment from the deleterious effects of excessive IL-1 production.

Aggregation of integrins and RhoA activation are required for Thy-1-induced morphological changes in astrocytes.

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a beta(3)-containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a beta(3)-containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC(1) astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominant-negative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein A-Sepharose beads. The same was shown by cross-linking beta(3)-containing integrin with anti-beta(3) antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on beta(3) integrin clustering and the resulting increase in RhoA activity.

Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion.

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse alpha to beta cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic beta cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of alpha to beta cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in beta cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.

Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro.

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

Antagonistic regulation of a proline-rich transcription factor by transforming growth factor beta and tumor necrosis factor alpha.

Transforming growth factor beta (TGF-beta) and tumor necrosis factor alpha (TNF-alpha) often exhibit antagonistic actions on the regulation of various activities such as immune responses, cell growth, and gene expression. However, the molecular mechanisms involved in the mutually opposing effects of TGF-beta and TNF-alpha are unknown. Here, we report that binding sites for the transcription factor CTF/NF-I mediate antagonistic TGF-beta and TNF-alpha transcriptional regulation in NIH3T3 fibroblasts. TGF-beta induces the proline-rich transactivation domain of specific CTF/NF-I family members, such as CTF-1, whereas TNF-alpha represses both the uninduced as well as the TGF-beta-induced CTF-1 transcriptional activity. CTF-1 is thus the first transcription factor reported to be repressed by TNF-alpha. The previously identified TGF-beta-responsive domain in the proline-rich transcriptional activation sequence of CTF-1 mediates both transcriptional induction and repression by the two growth factors. Analysis of potential signal transduction intermediates does not support a role for known mediators of TNF-alpha action, such as arachidonic acid, in CTF-1 regulation. However, overexpression of oncogenic forms of the small GTPase Ras or of the Raf-1 kinase represses CTF-1 transcriptional activity, as does TNF-alpha. Furthermore, TNF-alpha is unable to repress CTF-1 activity in NIH3T3 cells overexpressing ras or raf, suggesting that TNF-alpha regulates CTF-1 by a Ras-Raf kinase-dependent pathway. Mutagenesis studies demonstrated that the CTF-1 TGF-beta-responsive domain is not the primary target of regulatory phosphorylations. Interestingly, however, the domain mediating TGF-beta and TNF-alpha antagonistic regulation overlapped precisely the previously identified histone H3 interaction domain of CTF-1. These results identify CTF-1 as a molecular target of mutually antagonistic TGF-beta and TNF-alpha regulation, and they further suggest a molecular mechanism for the opposing effects of these growth factors on gene expression.