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Objective To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. Methods Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. Results In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. Conclusion These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.

Background Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. Objective We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. Methods Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. Results Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13–treated animals compared with levels seen in control animals. Conclusion These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. Clinical implications IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.

IL-13 contributes to airway hyperresponsiveness, mucus secretion, inflammation, and fibrosis, suggesting that it plays a central role in asthma pathogenesis. Neutralization of IL-13 with sIL-13Ralpha2-Fc (sIL-13R) reduces allergen-induced airway responses in rodent models of respiratory disease, but its efficacy in a large animal model has not been previously reported. In this study, we determined whether two different strategies for IL-13 neutralization modified experimental asthma in sheep. Sheep with natural airway hypersensitivity to Ascaris suum antigen were treated intravenously either with sIL-13R, a strong antagonist of sheep IL-13 bioactivity in vitro, or with IMA-638 (IgG1, kappa), a humanized antibody to human IL-13. Higher doses of IMA-638 were used because, although it is a potent antagonist of human IL-13, this antibody has 20 to 30 times lower binding and neutralization activity against sheep IL-13. Control animals received human IgG of irrelevant specificity. Sheep were treated 24 h before inhalation challenge with nebulized A. suum. The effects on antigen-induced early and late bronchial responses, and antigen-induced hyperresponsiveness, were assessed. Both sIL-13R and IMA-638 provided dose-dependent inhibition of the antigen-induced late responses and airway hyperresponsiveness. The highest dose of IMA-638 also reduced the early phase response. These findings suggest that IL-13 contributes to allergen-induced airway responses in this sheep model of asthma, and that neutralization of IL-13 is an effective strategy for blocking these A. suum-induced effects.

A novel class of 1H-(benzimidazol-2-yl)-1H-pyridin-2-one inhibitors of insulin-like growth factor I (IGF-1R) kinase is described. This report discusses the SAR of 4-(2-hydroxy-2-phenylethylamino)-substituted pyridones with improved IGF-1R potency.

The adhesion of a cell to its surrounding matrix is a key determinant in many aspects of cell behavior. Adhesion consists of distinct stages : attachment, cell spreading, motility, and/or immobilization. Interrelated signaling pathways regulate these stages, and many adhesion-related signals control the architecture of the cytoskeleton. The various cytoskeletal organizations then give rise to the specific stages of adhesion. It has been shown that arachidonic acid acts at a signaling branch point during cell attachment. Arachidonic acid is metabolized via lipoxygenase to activate actin polymerization and cell spreading. It is also metabolized by cyclooxygenase to generate small actin bundles. We have used confocal microscopy and indirect immunofluorescence to investigate the structure of these cyclooxygenase dependent actin bundles in HeLa cells. We have also employed cell migration assays and pharmacological modulation of cyclooxygenase and downstream signals. The results indicate that cyclooxygenase and PKA stimulate the formation of actin bundles that contain myosin II and associate with small focal adhesions. In addition, we demonstrate that this cytoskeletal organization correlates with increased cell motility.

Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma, parasite immunity, and tumor recurrence. At least two distinct receptor components, IL-4 receptor (R)α and IL-13Rα1, mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13, IL-13Rα2, whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Rα2, mice deficient in IL-13Rα2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Rα2−/− mice despite the fact that serum IL-13 was absent and immune interferon γ production increased compared with wild-type mice. IL-13Rα2–deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Rα2 in regulating immune responses in wild-type mice.

By using an immunoisolation procedure (Stan, R.-V., W.G. Roberts, K. Ihida, D. Predescu, L. Saucan, L. Ghitescu, and G.E. Palade. 1997. Mol. Biol. Cell. 8:595–605) developed in our laboratory, we have isolated a caveolar subfraction from rat lung endothelium and we have partially characterized the proteins of this subfraction which include an apparently caveolae-specific glycoprotein we propose to call PV-1 (formerly known as gp68). The isolation and partial sequencing of PV-1, combined with the cloning of the full length PV-1 cDNA led to the following conclusions: (a) PV-1 is a novel single span type II integral membrane protein (438 amino acids long) which forms homodimers in situ; (b) the transmembrane domain of PV-1 is near the NH2 terminus defining a short cytoplasmic endodomain and a large COOH-terminal ectodomain exposed to the blood plasma; (c) PV-1 is N-glycosylated and its glycan antennae bear terminal nonreducing galactosyl residues in α1-3 linkage. PV-1 is expressed mostly in the lung but both the messenger RNA and the protein can be detected at lower levels also in kidney, spleen, liver, heart, muscle, and brain. No signal could be detected in testis and two lower molecular weight forms were detected in brain. Immunocytochemical studies carried out by immunodiffusion on rat lung with an anti–PV-1 polyclonal antibody directed against a COOH-terminal epitope reveal a specific localization of PV-1 to the stomatal diaphragms of rat lung endothelial caveolae and confirm the extracellular orientation of the PV-1 COOH terminus.

A monoclonal antibody raised against purified rat lung endothelial plasma membranes was found to recognize an apparently novel, 85 kD, integral endothelial plasma membrane glycoprotein. By immunofluorescence and electron microscope immunocytochemistry, this endothelial antigen was detected at the luminal and tissue fronts of all rat endothelia, except those of discontinuous type (liver and spleen sinusoids). In lung alveolar capillaries the antigen appeared to be uniquely associated with the very attenuated endothelial processes forming the blood-air barrier, and virtually absent on the surface of the rest of the cell, where the nucleus and the organelles are located. No other cells, except fibroblasts, appeared to be labeled by this monoclonal antibody.

Summary: MRL/lpr (Fas-deficient) mice develop an autoimmune syndrome associated with excessive production of autoantibodies. A significant portion of these autoantibodies are IgG2a molecules specific for many of the autoantigens recognized by the sera of patients with systemic lupus erythematosus. In addition, MRL/lpr mice make exceedingly high titers of IgG or IgA rheumatoid factors (RF) specific for autologous IgG2a, The microenvironment of the IgG2a-producing B cells as well as the prototypic RF autoantibodies was determined by a combination of immunohistochemical and m situ hybridization techniques. In contrast to the anti body-producing cells present in mice responding to conventional foreign antigens, both IgG2a+ and RF+ B cells were found to be densely clustered in the T-cell-rich inner periarteriolar lymphatic sheath of the spleen. These results suggest that conventional antibody and autoantibody production in MRL/lpr mice may be mechanistically distinct processes.

A major mechanism maintaining immune tolerance is the deletion of potentially autoreactive thymocytes by apoptosis during development in the thymus. Previous reports suggest that apoptosis is induced by high avidity signals transduced via the T cell receptor; however, the role of signals transduced by other cell surface receptors during thymic selection remains poorly understood. Fas, a member of the TNF receptor family, has been shown to induce apoptosis in mature peripheral T cells; however, the effects of Fas on negative selection of thymocytes have not been previously detected. Using a sensitive terminal deoxynucleotidyl transferase method to detect apoptotic cells, we found that mutant Fas molecules in Ipr mice decrease the sensitivity of thymocytes to T cell receptor-mediated apoptosis and that blockade of Fas-Fas ligand interactions in vivo can inhibit antigen-induced apoptosis of thymocytes in non-Ipr mice. Thus, we have shown that Fas, in conjunction with antigen-specific signals, can modulate apoptosis during negative selection of thymocytes.

The goal of this study was to examine the in vivo site of autoantibody production in normal and autoimmune-prone mice. B cells were identified in tissue sections with IgM- and IgG2a-specific riboprobes that readily distinguished resting cells from antibody-forming cells (AFC). In normal mice, the few identifiable IgG2a-secreting cells were found in the red pulp. By contrast, in Ipr mice exceedingly high numbers of IgG2a and autoantibody-producing cells were found deep within the T cell-rich periarteriolar lymphoid sheaths (PALS). This unusual anatomic location of autoantibody-secreting B cells is unique to Fas dysregulated strains, since IgG2a-producing cells in MRL/+ and (SWR × NZB)F1 mice were found predominantly in the red pulp or outer PALS, similar to normal mice. Furthermore, analysis of spleens from Ipr and non- Ipr anti-DNA immunoglobulin transgenic mice revealed dramatic accumulation of Tg + cells in the inner PALS only in Ipr mice. These data suggest that in the absence of Fas, autoreactive B cells accumulate in T cell-rich zones, and this anatomic feature may contribute to autoantibody production.

The ability to overexpress [2Fe-2S] ferredoxins in Escherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegatative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in in these proteins in their oxidized and reduced states has been studied by 1H, 2H, 13C, and 15N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).

Immunization of the autoimmune mouse strain (M × A) Id/lpr with Ars-KLH, has been shown to elicit a prolonged anti-Ars IdCR response similar to that found in A/J mice. Cell fusion of splenocytes from a diseased mouse previously immunized with Ars-KLH resulted in a monoclonal antibody, 1–52.30, that was found to express the strain A major cross-reactive idiotype, but failed to bind Ars. Nucleotide sequence analysis demonstrated that 1–52.30: (a) used the “canonical” combination of gene segments associated with this idiotype, and (b) exhibited a pattern of somatic mutation consistent with selection for high affinity Ars binding. Two amino acids, VL 91 and 93, were mutated in 36–65, the germline equivalent of the IdCR antibodies, to 1–52.30-like residues (91G → D, 93T → M). The results of the mutagenesis showed that changing a single light chain residue, VL 91, from glycine to aspartic acid, resulted in a dramatic loss of Ars binding activity.

In this report we describe a rapid, high-yield protocol for the isolation of apical (AP) and basolateral (BL) plasma membrane domains from monolayers of bovine aortic endothelial cells (BAECs) grown on tissue culture dishes as well as microcarrier beads. Using a modified cationic colloidal silica microbead membrane-isolation procedure, which deposits a uniform silica-polyacrylate pellicle over the entire AP membrane surface, a 4- to 9.6-fold relative enrichment of AP membrane and a 3.55- to 3.67-fold relative enrichment of BL membrane was obtained when the isolated domains were examined for silica and Na+/K(+)-ATPase, respectively. Immunoblotting of the isolated membrane domains displayed the presence of angiotensin-converting enzyme (ACE) exclusively in the AP domain and collagen receptors (CRs) highly enriched in the BL membrane domain when monolayers were grown on a gelatin substratum.

This project examines the transcellular membrane protein polarity of bovine aortic endothelial cell (BAEC) monolayers in vitro with respect to the roles that intercellular junctions (as defined by comparing confluent and subconfluent monolayers) and the submembranous cytoskeleton play in controlling this phenomenon. Plasma membrane (PM) proteins obtained from apical (AP) and basolateral (BL) PM domains of confluent BAEC monolayers were isolated using the cationic colloidal silica technique and resolved by two-dimensional gel electrophoresis (2-D PAGE). To facilitate the identification of domain-specific PM proteins, an isoelectric point/molecular weight database of the proteins from AP and BL PM domains was constructed. Domain-specific PM proteins were assessed for their interaction with the cytoskeleton by determining whether they co-isolated with a Triton X-100 detergent-resistant cytoskeletal/extracellular matrix fraction. The maintenance of polarized PM protein segregation by intercellular junctional complexes was determined by comparing AP and BL protein patterns of confluent monolayers with patterns generated by subconfluent monolayers, which lack such junctional structures. Proteins isolated from AP and BL PM domains from both confluent states were immunoblotted with antibodies to angiotensin-converting enzyme (ACE) and collagen receptors (CR). ACE was restricted exclusively to the AP PM domain in the subconfluent condition, even though no apparent cytoskeletal interaction was observed. CRs, found to interact with the cytoskeleton in either confluence state, were predominantly segregated to the BL PM domain regardless of the presence or absence of cell-cell contact. Membrane proteins found by 2-D PAGE to be asymmetrically distributed in the absence of intercellular junctions were assessed for cytoskeletal interaction by their inability to be extracted by Triton X-100 from monolayers in the subconfluent state. Computer cross-referencing of 2-D PAGE peak lists and immunodetection generated from the above fractionation protocols identifies a set of four proteins associated with the cytoskeleton that remain segregated in the proper domain, and five proteins associated with the cytoskeleton that become equally distributed between AP and BL PM domains in the absence of intercellular junctions. Additionally, six proteins not associated with the cytoskeleton remain asymmetrically distributed to the AP domain in the subconfluent state. The data suggest that BAEC monolayers have unknown mechanisms, apart from intercellular junctions expressed at confluency or cytoskeletal binding, for maintaining transcellular PM protein polarity.

The solution properties of two fluorescent lipophilic analogues were examined in conjunction with their ability to penetrate the tight junctions of bovine aortic endothelial cell monolayers. 5-(N-dodecanoyl)aminofluoroscein was shown to label both the apical and basolateral plasma membrane domains of confluent monolayers at 4 degrees C and pH 7.3, but 5-(N-hexadecanoyl)aminofluoroscein was shown to label only the apical membrane domain. When used under more soluble conditions at 20 degrees C and pH 8.5, both probes labeled apical and basolateral plasma membrane domains more equally. This indicates that solubility conditions, and not tight junctions, dictate the penetration of 5-(N-hexadecanoyl)aminofluoroscein from the apical to the basolateral plasma membrane domain.

Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of 35S-methionine-labeled whole cells. Two-dimensional gel analysis of 35S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture. Furthermore, expression of specific subsets of cell surface proteins is dependent on environmental conditions, and in some cases is both cell-type and media-type dependent. Thus, even though endothelial cells are considered terminally differentiated cells, there exists additional or "latent" heterogeneity in the ability of these different cells to respond to "developmental signals" (i.e. mitogenic medium) in vitro.

The structure of the complex formed between bovine beta-trypsin and the highly potent synthetic inhibitor 2-{3'-[5'-methoxy-2'-(N-p-diaminomethylphenyl)amido]-1'-pyrido}-5-(N-2''-t-butylethanol)amidobenzoic acid (C(28)H(32)N(5)O(6)) has been determined at 0.97 A resolution. X-ray intensity data were collected to 0.97 A under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX-97 to R(cryst) factors of 13.4 and 12.6% and R(free) factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |F(o) - F(c)| difference map, which accounts for an estimated 35% of all H atoms at the 2.5sigma level. The C, N and O atoms are definitively differentiated in the electron-density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand-binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195-His57-Asp102, thereby blocking the proton-relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 N(delta1) and Asp102 O(delta1).

Transparent substrates having heterogeneities ranging from nanometer to micrometer lateral length scale were fabricated to study cell migration. The surfaces were generated using thin films of block copolymers and homopolymer blends on ultra smooth transparent polyethylene terephthalate films. Results show that the lateral size scale of the surface heterogeneities affects fibroblast (NIH-3T3) adhesion, spreading and motility. More specifically, fibroblasts migrate faster on micron-sized than on nanometer-sized heterogeneities. Cell movements and morphology on the micron patterned surfaces resemble cells cultured in a 3D environment. These surfaces, therefore, can potentially be utilized as models to study cell behavior in physiologically relevant conditions which can add to our fundamental understanding of cell-substrate interactions and facilitate development of surfaces for medical devices.

Polymeric substrates of different surface chemistry and length scales were found to have profound influence on cell adhesion. The adhesion of fibroblasts on surfaces of oxidized polystyrene (PS), on surfaces modified with random copolymers of PS and poly(methyl methacrylate) [P(S-r-MMA)] with topographic features, and chemically patterned surfaces that varied in lateral length scales from nanometers to microns were studied. Surfaces with heterogeneous topographies were generated from thin film mixtures of a block copolymer, PS-b-MMA, with homopolymers of PS and PMMA. The two homopolymers macroscopically phase separated and, with the addition of diblock copolymer, the size scales of the phases decreased to nanometer dimensions. Cell spreading area analysis showed that a thin film of oxidized PS surface promoted adhesion whereas a thin film of P(S-r-MMA) surface did not. Fibroblast adhesion was examined on surfaces in which the lateral length scale varied from 60 nm to 6 μm. It was found that, as the lateral length scale between the oxidized PS surfaces decreased, cell spreading area and degree of actin stress fiber formation increased. In addition, scanning electron microscopy was used to evaluate the location of filopodia and lamellipodia. It was found that most of the filopodia and lamellipodia interacted with the oxidized PS surfaces. This can be attributed to both chemical and topographic surface interactions that prevent cells from interacting with the P(S-r-MMA) at the base of the topographic features. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 462–469, 2004

Wound healing involves multiple cell signaling pathways, including those regulating cell-extracellular matrix adhesion. Previous work demonstrated that arachidonate oxidation to leukotriene B4 (LTB4) by 5-lipoxygenase (5-LOX) signals fibroblast spreading on fibronectin, whereas cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) formation facilitates subsequent cell migration. We investigated arachidonate metabolite signaling in wound closure of perturbed NIH/3T3 fibroblast monolayers. We found that during initial stages of wound closure (0–120 min), all wound margin cells spread into the wound gap perpendicularly to the wound long axis. At regular intervals, between 120 and 300 min, some cells elongated to project across the wound and meet cells from the opposite margin, forming distinct cell bridges spanning the wound that act as foci for later wound-directed cell migration and resulting closure. 5-LOX inhibition by AA861 demonstrated a required LTB4 signal for initial marginal cell spreading and bridge formation, both of which must precede wound-directed cell migration. 5-LOX inhibition effects were reversible by exogenous LTB4. Conversely, COX inhibition by indomethacin reduced directed migration into the wound but enhanced early cell spreading and bridge formation. Exogenous PGE2 reversed this effect and increased cell migration into the wound. The differential effects of arachidonic acid metabolites produced by LOX and COX were further confirmed with NIH/3T3 fibroblast cell lines constitutively over- and underexpressing the 5-LOX and COX-2 enzymes. These data suggest that two competing oxidative enzymes in arachidonate metabolism, LOX and COX, differentially regulate sequential aspects of fibroblast wound closure in vitro.

IL-13 is a Th2 cytokine that plays crucial roles in the pathophysiology of allergy, asthma and helminth infection. The high affinity receptor for IL-13, IL-13Ralpha2, may act as a decoy receptor for IL-13. The anti-tumor effect of IL-13 and its soluble receptor IL-13Ralpha2 have been examined in different tumor systems. Previous studies have shown that IL-13 enhances anti-tumor responses in some model systems, whereas IL-13Ralpha2Fc prevents IL-13 mediated suppression of tumor immuno-surveillance in a different model system. In this study, we have used a cytokine (receptor) gene therapy approach and studied the immune responses mediated by IL-13 and IL-13Ralpha2Fc in poorly immunogenic B16F1 melanoma and immunogenic MethA fibrosarcoma tumor models. We find that IL-13 reduces the tumorigenicity of B16F1 melanoma and MethA fibrosarcoma cells in vivo, most likely through the recruitment of neutrophils and macrophages. IL-13 mediated anti-tumor responses do not lead to the generation of tumor-specific T cells. Neither IL-13Ralpha2Fc gene transduction nor in vivo treatment with soluble IL-13Ralpha2Fc has a statistically significant effect of tumor growth. IL-13Ralpha2 deficient host background does not alter tumor growth, suggesting that endogenous levels of IL-13 do not contribute to an anti-tumor response in these models. We conclude that IL-13, but not soluble IL-13Ralpha2, has anti-tumor activity in the models described here, possibly by enhancing innate anti-tumor immunity.

Cell adhesion to the extracellular matrix via integrins is a primary regulatory mechanism for numerous aspects of normal cellular function. However, disruption of this interaction can result in pathology. For example, one characteristic of transformed cells is loss of adhesion dependence for viability. Adhesion also is a necessary step in tumor metastasis. It has been shown previously, in HeLa cells, that cell attachment to a gelatin-coated substrate results in the release of arachidonic acid, which is metabolized by lipoxygenase. A subsequent cascade of lipid second messengers activates protein kinase C, which triggers actin polymerization leading to cell spreading. We now demonstrate by inhibitor studies and biochemical analysis, a parallel branch of arachidonic acid signaling that reorganizes the actin cytoskeleton into small bundles. This branch of the pathway is initiated by cyclooxygenase, which generates prostaglandins and causes the downstream activation of cyclic AMP-dependent protein kinase. This work elucidates a system of interacting signals in which arachidonic acid functions at a branch point in cytoskeletal signaling. The lipoxygenase branch provides polymerized actin; these actin filaments act as a substrate for the cylooxygenase branch to generate actin bundles. Cell Motil. Cytoskeleton 53:239–250, 2002. © 2002 Wiley-Liss, Inc.

Adhesion of cells to an extracellular matrix is characterized by several discrete morphological and functional stages beginning with cell-substrate attachment, followed by cell spreading, migration, and immobilization. We find that although arachidonic acid release is rate-limiting in the overall process of adhesion, its oxidation by lipoxygenase and cyclooxygenases regulates, respectively, the cell spreading and cell migration stages. During the adhesion of NIH-3T3 cells to fibronectin, two functionally and kinetically distinct phases of arachidonic acid release take place. An initial transient arachidonate release occurs during cell attachment to fibronectin, and is sufficient to signal the cell spreading stage after its oxidation by 5-lipoxygenase to leukotrienes. A later sustained arachidonate release occurs during and after spreading, and signals the subsequent migration stage through its oxidation to prostaglandins by newly synthesized cyclooxygenase-2. In signaling migration, constitutively expressed cyclooxygenase-1 appears to contribute ∼25% of prostaglandins synthesized compared with the inducible cyclooxygenase-2. Both the second sustained arachidonate release, and cyclooxygenase-2 protein induction and synthesis, appear to be regulated by the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK)1/2. The initial cell attachment-induced transient arachidonic acid release that signals spreading through lipoxygenase oxidation is not sensitive to ERK1/2 inhibition by PD98059, whereas PD98059 produces both a reduction in the larger second arachidonate release and a blockade of induced cyclooxygenase-2 protein expression with concomitant reduction of prostaglandin synthesis. The second arachidonate release, and cyclooxygenase-2 expression and activity, both appear to be required for cell migration but not for the preceding stages of attachment and spreading. These data suggest a bifurcation in the arachidonic acid adhesion-signaling pathway, wherein lipoxygenase oxidation generates leukotriene metabolites regulating the spreading stage of cell adhesion, whereas ERK 1/2-induced cyclooxygenase synthesis results in oxidation of a later release, generating prostaglandin metabolites regulating the later migration stage.

Inhibition of PLA2 activity and rescue by addition of exogenous AA was used to demonstrate that AA production is essential for integrin-mediated NIH-3T3 murine cell spreading. Both AA release and cell spreading after attachment to a FN substrate were inhibited by the PLA2 inhibitor mepacrine. AA release was essential for signaling spreading since the inhibition of spreading induced by mepacrine was overcome by exogenous AA. Cells ectopically expressing full-length chicken beta1-integrins both released AA and spread fully on a substrate of anti-chicken beta1-integrin monoclonal antibody, and inhibition of PLA2 by mepacrine suppressed both spreading and AA release. Exogenous AA also reversed this mepacrine-induced inhibition of spreading. The role of the beta1-integrin cytosolic domain in AA release was examined by comparing responses of cells expressing full-length chicken beta1-integrins versus cells expressing a deletion mutant chicken beta1-integrin with a truncated cytosolic domain. Cells expressing a truncated chicken beta1-integrin released significantly less AA and failed to spread on the anti-chicken beta1-integrin antibody substrate. Furthermore, clustering full-length receptors with soluble antibody stimulated greater AA release than clustering of receptors having truncated cytosolic domains. These data suggest the beta1-integrin cytosolic domain is required for optimal PLA2 activation to produce AA necessary for cell spreading.

MRL/lpr mice spontaneously develop a lupus-like autoimmune syndrome characterized by immunopathologic manifestations such as necrotizing vasculitis of the skin and glomerulonephritis. A feature of this autoimmune syndrome is the production of extremely large amounts of monoclonal IgG3 cryoglobulins. The structural basis of IgG3 cryoprecipitation is not well understood. Although the IgG3 isotype is necessary for cryoprecipitation, not all IgG3 antibodies cryoprecipitate. It has been postulated that electrostatic charge may be influential in cryoprecipitation. To investigate this problem, the VH and VL sequences of a panel of IgG3 cryoglobulins and non-cryoglobulins were compared, with particular attention to charged amino acid differences. At VH residues 6 and 23 the cryoglobulins were more positively charged than their non-cryoglobulin counterparts. To analyze further the effect of charge on cryoprecipitation, the sequence of an IgG3 monoclonal cryoprecipitating rheumatoid factor was modified by site-directed mutagenesis. The more positive residues at VH 6 and 23 present in some of the cryoglobulin antibodies were mutated to the more negative residues found in the non-cryoglobulins. The results show that VH residue 6 affects cryoprecipitation while residue 23 does not. When injected into normal BALB/c mice, the unmutated antibody produced glomerular immune deposits and focal glomerulonephritis, whereas loss of cryoprecipitability by mutating residue 6 completely abrogated glomerular immune deposition and glomerular injury. In contrast, the mutation at residue 23 which retains cryoprecipitability reduced glomerular immune deposition and prevented glomerular injury.

The three-dimensional structure of the fatty-acid-binding protein isolated from the flight muscle of the desert locust Schistocerca gregaria has been solved and refined to a crystallographic R-value of 18.5% for all measured X-ray data from 30.0- to 2.2-A resolution. Crystals employed in the investigation were grown from 2.6 to 2.8 M ammonium sulfate solutions, buffered at pH 7.5 and containing 2-5% 2-methyl- 2,4-pentanediol. They belonged to the space group P2, with unit cell dimensions of a = 61.6 A, b = 44.8 A, c = 63.9 A, and /3 = 113.6O and two molecules per asymmetric unit. The protein fold consists of ten strands of antiparallel P-pleated sheet that wrap around to form a B-barrel. In addition, there are two small a-helices and six type I, two type 11, and two type 11' turns. The two molecules pack in the asymmetric unit as a dimer with a local 2-fold rotational axis. The subunit-subunit interface involves amino acid side chains located in the area of the helix-turn-helix motif and the turn between &strands E and F. It is this area that has been speculated to form the portal through which fatty acids enter the binding cavity. There are 23 solvent molecules that are conserved between the two independent molecules in the asymmetric unit. Nine of these waters play important structural roles. A three-dimensional comparison between the insect and human muscle fatty-acid-binding proteins shows that their a-carbons superimpose with a root-mean- square deviation of 0.77 A for 89 structurally equivalent atoms. This high tertiary homology between these two proteins is surprising in light of the fact that they developed independently for more than 500 million years.

Smyth line (SL) chickens are phenotypically characterized by a posthatch depigmentation (vitiligo) of the feathers. The destruction of melanocytes in the feather follicle as well as in other tissues such as the choroid is genetically determined. Previous studies have shown that bursectomy or treatment with immunosuppressive agents decreases the incidence and severity of SL depigmentation (1). These observations implicate a role for the immune system, specifically the humoral component, in melanocyte destruction. In this study we show that there are circulating melanocyte-specific autoantibodies in the sera of depigmented SL chicks which are not present in sera from Light Brown Leghorn (LBL) control chicks. By immunoblots and by immunoprecipitation of radiolabeled melanocyte proteins, SL autoantibodies were shown to bind to multiple melanocyte proteins between 65 and 80 kDa. These proteins are not detected in SL fibroblasts. By immunoblotting, the incidence of autoantibodies for these 65- to 80-kDa proteins was determined to be 95% in depigmented SL chicks (n = 20), 0% in normally pigmented SL chicks (n = 8), and 5% in LBL chicks (n = 20). Melanocyte autoantibodies are detectable in the sera of affected chicks at or several weeks prior to the expression of depigmentation. This information, plus previously published data, implicate melanocyte autoantibodies in the depigmentary phenomenon of vitiligo observed in Smyth line chickens.

T lymphocytes from aged donors function poorly, but the biochemical basis for the defect remains uncertain. We tested the hypothesis that T cells from old mice had a diminished ability to transmit extracellular signals into the cytoplasm, by measuring intracellular free calcium concentrations (Cai) in T cells stimulated by the polyclonal activator concanavalin A (Con A). Using the second-generation fluorochrome indo-1 as a reporter of Cai, we found that the Con A-induced elevation of Cai levels is reduced both in rate and extent in old T cells, as compared to T cells from young mice. Flow cytometric analysis showed that this age-sensitive change represents a decline, with age, in the number of T cells that can respond to Con A by increasing their Cai above resting baseline levels (100-120 nM). These results thus show that defects in activation are manifested by T cells from old donors within the first 5 minutes of the activation process, and suggest that aging may lead to alterations either in the surface molecules that receive extracellular signals, or in the sequence of coupled events by which these extracellular signals bring about alterations in the intracellular ionic milieu.

Concentrations of concanavalin A that induced patching and capping of cell surface receptors on Dictyostelium discoideum also induce binding of the receptors to the cortical cytoskeleton, which was isolated by density-gradient centrifugation. The receptors were solubilized by deoxycholate, purified by affinity chromatography, and used to determine whether the receptors bound directly to the cytoskeletal protein, actin. As the concentration of actin was increased, many of the receptors became bound to purified filamentous rabbit muscle actin, even in the absence of concanavalin A. As in the ligation-induced binding of receptors to the cortical cytoskeleton in cells, concanavalin A induced much stronger binding of the purified receptors to filamentous actin. The results were consistent with a previously stated hypothesis that induction of receptor binding to the cytoskeleton during their patching and capping is driven by clustering the receptors, which reduces their translational entropy and by doing so enhances their avidity for the cytoskeleton.

Cytoplasmic calcium concentration ([Ca]i) rises within minutes of exposure of T lymphocytes to a mitogen. T cells from old mice are defective in this reaction, a defect that could reflect either altered signal transduction or instead a more general age-associated change in intracellular calcium regulation. We therefore tested the ability of T cells from old mice to regulate their [Ca]i concentration after exposure to low concentrations of ionomycin, an agent that raises [Ca]i but bypasses receptor-mediated signal transduction mechanisms. Exposure of T cells to ionomycin leads to an abrupt increase in [Ca]i followed by stabilization at a dose-dependent plateau level that is affected by extracellular EGTA, by calmodulin inhibitors, and by modulators of protein kinase C. Plateau levels of [Ca]i after ionomycin challenge were consistently lower in T cells from old mice than in T cells from young mice. Flow cytometric experiments showed that while essentially all T cells from both old and young mice responded to ionomycin, they did so to an extent that depended on donor age. The age-dependent increase in resistance to ionomycin-induced changes in [Ca]i cannot be attributed to diminished membrane permeability to the ionomycin-calcium complex. The data suggest that aging may lead, in T lymphocytes, to a relative resistance to increases in [Ca]i, a resistance that in turn prevents cell activation.

A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S3 cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally.

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells, grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins absorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.

Carbon dioxide from untreated and alloxanized rats was collected free of atmospheric CO2 and then analyzed for its stable carbon isotope (13C12C) ratio. Alloxan induced diabetic rats expired CO2 with a 13C12C ratio less than that for CO2 collected from untreated rats. The difference in 13C12C ratios indicated that alloxanized rats oxidized more lipid than control rats since rat lipid has a 13C12C ratio less than that of glycogen.

Two types of polyethylenimine-coated glass microbeads (13-44 micrometer) were synthesized and used for the immobilization of glucose oxidase from Aspergillus niger and catalase from A. niger and beef liver. The two types of beads were distinguishable by differences in their surface topography. Immobilizations were performed by adsorption followed by treatment with glutaraldehyde. The immobilized-enzyme activities per unit support of all of the enzymes tested were compared with and found to be superior to the immobilized activities attainable on aminopropyl-activated glass microbeads. When enzyme was present in less than saturating amounts, the coated beads were able to remove 100% of the glucose oxidase activity initially present in the immobilization solution, with 78-87% of that activity expressed on the support surface. Bound glucose oxidase was more stable to thermal inactivation than native enzyme.