Bristol-Myers Squibb (Japan)

BACKGROUND: Effective new therapies are needed for rheumatoid arthritis. Current therapies target the products of activated macrophages; however, T cells also have an important role in rheumatoid arthritis. A fusion protein--cytotoxic T-lymphocyte-associated antigen 4-IgG1 (CTLA4Ig)--is the first in a new class of drugs known as costimulation blockers being evaluated for the treatment of rheumatoid arthritis. CTLA4Ig binds to CD80 and CD86 on antigen-presenting cells, blocking the engagement of CD28 on T cells and preventing T-cell activation. A preliminary study showed that CTLA4Ig may be effective for the treatment of rheumatoid arthritis. METHODS: We randomly assigned patients with active rheumatoid arthritis despite methotrexate therapy to receive 2 mg of CTLA4Ig per kilogram of body weight (105 patients), 10 mg of CTLA4Ig per kilogram (115 patients), or placebo (119 patients) for six months. All patients also received methotrexate therapy during the study. The clinical response was assessed at six months with use of the criteria of the American College of Rheumatology (ACR), which define the response according to its extent: 20 percent (ACR 20), 50 percent (ACR 50), or 70 percent (ACR 70). Additional end points included measures of the health-related quality of life. RESULTS: Patients treated with 10 mg of CTLA4Ig per kilogram were more likely to have an ACR 20 than were patients who received placebo (60 percent vs. 35 percent, P<0.001). Significantly higher rates of ACR 50 and ACR 70 responses were seen in both CTLA4Ig groups than in the placebo group. The group given 10 mg of CTLA4Ig per kilogram had clinically meaningful and statistically significant improvements in all eight subscales of the Medical Outcomes 36-Item Short-Form General Health Survey. CTLA4Ig was well tolerated, with an overall safety profile similar to that of placebo. CONCLUSIONS: In patients with active rheumatoid arthritis who were receiving methotrexate, treatment with CTLA4Ig significantly improved the signs and symptoms of rheumatoid arthritis and the health-related quality of life. CTLA4Ig is a promising new therapy for rheumatoid arthritis.

BACKGROUND: Renal transplantation is the standard of care for patients with end-stage renal disease. Although maintenance immunosuppression with calcineurin inhibitors yields excellent one-year survival, it is associated over the long term with high rates of death and graft loss, owing in part to the adverse renal, cardiovascular, and metabolic effects of these agents. The use of potentially less toxic agents, such as belatacept, a selective blocker of T-cell activation, may improve outcomes. METHODS: We randomly assigned renal-transplant recipients to receive an intensive or a less-intensive regimen of belatacept or cyclosporine. All patients received induction therapy with basiliximab, mycophenolate mofetil, and corticosteroids. The primary objective was to demonstrate the noninferiority of belatacept over cyclosporine in the incidence of acute rejection at six months (with an upper bound of the 95 percent confidence interval around the treatment difference of less than 20 percent). RESULTS: At six months, the incidence of acute rejection was similar among the groups: 7 percent for intensive belatacept, 6 percent for less-intensive belatacept, and 8 percent for cyclosporine. At 12 months, the glomerular filtration rate was significantly higher with both intensive and less-intensive belatacept than it was with cyclosporine (66.3, 62.1, and 53.5 ml per minute per 1.73 m2, respectively), and chronic allograft nephropathy was less common with both regimens of belatacept than with cyclosporine (29 percent, 20 percent, and 44 percent, respectively). Lipid levels and blood-pressure values were similar or slightly lower in the belatacept groups, despite the greater use of lipid-lowering and antihypertensive medications in the cyclosporine group. CONCLUSIONS: Belatacept, an investigational selective costimulation blocker, did not appear to be inferior to cyclosporine as a means of preventing acute rejection after renal transplantation. Belatacept may preserve the glomerular filtration rate and reduce the rate of chronic allograft nephropathy.

UNLABELLED: All-oral combinations of direct-acting antivirals may improve efficacy and safety outcomes for patients with hepatitis C virus (HCV) infection, particularly those who are poor candidates for current interferon/ribavirin-based regimens. In this open-label, phase 3 study, 135 interferon-ineligible/intolerant and 87 nonresponder patients with chronic HCV genotype 1b infection were enrolled at 24 centers in Japan. Patients received daclatasvir 60 mg once daily plus asunaprevir 100 mg twice daily for 24 weeks. The primary endpoint was sustained virologic response 24 weeks after treatment (SVR24 ). This study is registered with ClinicalTrials.gov (NCT01497834). SVR24 was achieved by 87.4% of interferon-ineligible/intolerant patients and 80.5% of nonresponder (null and partial) patients; rates were similar in cirrhosis (90.9%) and noncirrhosis (84.0%) patients, and in patients with IL28B CC (84.5%) or non-CC (84.8%) genotypes. Fourteen patients in each group (12.6%) discontinued dual therapy, mainly due to adverse events or lack of efficacy. Nine nonresponder patients received additional treatment with peginterferon/ribavirin per protocol-defined criteria. The rate of serious adverse events was low (5.9%) and varied among patients. The most common adverse events were nasopharyngitis, increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST), headache, diarrhea, and pyrexia. CONCLUSION: Interferon-free, ribavirin-free all-oral therapy with daclatasvir and asunaprevir for 24 weeks is well tolerated and can achieve a high rate of SVR in patients with HCV genotype 1b who were ineligible, intolerant, or had not responded to prior interferon-based therapy. (Hepatology 2014;59:2083-2091).

Intermolecular multiple-quantum coherences between bulk water and a glycoprotein fragment at modest concentration (20 mM) have been experimentally produced and detected, although such coherences are inconceivable in the normal theoretical framework of nuclear magnetic resonance. A density matrix treatment explains these results by including the long-range dipolar interaction between spins and by discarding the high-temperature approximation. These results imply that peak intensities (critical for structural determinations) can be distorted in many gradient experiments, and show that magic-angle gradients provide substantial improvements with reduced gradient strengths. They also suggest methods for contrast enhancement in magnetic resonance imaging.

OBJECTIVE: Oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine (oxPAPC) accumulates in atherosclerotic lesions and in vitro studies suggest that it mediates chronic inflammatory response in endothelial cells (ECs). The goal of our studies was to identify pathways mediating the induction of inflammatory genes by oxPAPC. METHODS AND RESULTS: Using expression arrays, quantitative polymerase chain reaction (PCR), and immunoblotting we demonstrate that oxPAPC leads to endoplasmic reticulum stress and activation of the unfolded protein response (UPR) in human aortic ECs. Immunohistochemistry analysis of human atherosclerotic lesions indicated that UPR is induced in areas containing oxidized phospholipids. Using the UPR inducing agent tunicamycin and selective siRNA targeting of the ATF4 and XBP1 branches of the UPR, we demonstrate that these transcription factors are essential mediators of IL8, IL6, and MCP1 expression in human aortic ECs required for maximal inflammatory gene expression in the basal state and after oxPAPC treatment. We also identify a novel oxPAPC-induced chemokine, the CXC motif ligand 3 (CXCL3), and show that its expression requires XBP1. CONCLUSIONS: These data suggest that the UPR pathway is a general mediator of vascular inflammation and EC dysfunction in atherosclerosis, and, likely, other inflammatory disorders.

UNLABELLED: Patients with chronic hepatitis C virus (HCV) infection and previous null response to pegylated interferon (Peg-IFN) and ribavirin (RBV) have limited therapeutic options. HCV genotype 1 is the most common worldwide and the most difficult to treat; genotype 1b is the most common subtype of genotype 1 outside North America. The enhanced antiviral activity achieved by combining two direct-acting antiviral (DAA) agents may improve clinical outcomes. This open-label, phase IIa study included 10 patients with chronic HCV genotype 1b infection and previous null response (<2 log(10) reduction in HCV RNA after 12 weeks) to Peg-IFN and RBV. Patients received dual DAA treatment for 24 weeks with the nonstructural protein 5A replication complex inhibitor, daclatasvir (60 mg once-daily), and the nonstructural protein 3 protease inhibitor, asunaprevir (initially 600 mg twice-daily, then subsequently reduced to 200 mg twice-daily). The primary efficacy endpoint was the proportion of patients with sustained virologic response (SVR) at 12 weeks post-treatment (SVR(12) ). Nine patients completed 24 weeks of treatment; 1 patient discontinued treatment after 2 weeks. In the 9 patients who completed the full course of treatment, HCV RNA was undetectable at week 8 and remained undetectable through the end of treatment; all 9 patients achieved SVR(12) and SVR(24) . HCV RNA also remained undetectable post-treatment in the patient who discontinued after 2 weeks. There was no viral breakthrough. Diarrhea and headache, generally mild, were the most common adverse events; transaminase elevations were reported in 3 patients, but did not result in discontinuation. CONCLUSIONS: Dual therapy with daclatasvir and asunaprevir, without Peg-IFN and RBV, can achieve high SVR rates in difficult-to-treat patients with HCV genotype 1b infection and previous null response to Peg-IFN and RBV.

Effects of oral administration of the angiotensin II receptor antagonist (selective AT(1)-subtype) irbesartan on glucose tolerance and insulin action on skeletal-muscle glucose transport were assessed in the insulin-resistant obese Zucker rat. In the acute study, obese rats received either vehicle (water) or irbesartan 1 hour before the experiment. Although irbesartan had no effect on glucose transport (2-deoxyglucose uptake) in the epitrochlearis muscle, which consists mainly of type IIb fibers, acute angiotensin II receptor antagonism led to a dose-dependent increase in insulin action in the predominantly type I soleus muscle. Irbesartan at 25 and 50 mg/kg induced significant increases (41% and 50%, respectively; P<0.05) in insulin-mediated glucose transport. Moreover, these acute irbesartan-induced improvements in soleus-muscle glucose transport were associated with enhancements in whole-body insulin sensitivity (r=-0.732; P<0.05), as assessed during an oral glucose tolerance test. After chronic administration of irbesartan (21 days at 50 mg. kg(-1). d(-1)), glucose tolerance was enhanced further, and insulin-mediated glucose transport was significantly elevated in both epitrochlearis (32%) and soleus (73%) muscle. Chronic angiotensin II receptor antagonism was associated with significant increases in glucose transporter-4 (GLUT-4) protein expression in soleus (22%) and plantaris (20%) muscle and myocardium (15%). Chronic irbesartan-induced increases in whole-body insulin sensitivity were associated with increased insulin-mediated glucose transport in both epitrochlearis (r=-0.677; P<0.05) and soleus (r=-0.892; P<0.05) muscle. In summary, angiotensin II receptor (AT(1)-subtype) antagonism, either acutely or chronically, improves glucose tolerance, at least in part because of an enhancement in skeletal-muscle glucose transport, and the effect of chronic angiotensin II receptor antagonism on type I skeletal-muscle glucose uptake is associated with an increase in GLUT-4 protein expression.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor-deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO-overexpressing, LDL receptor-deficient mice (LDLR-/-/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor-deficient (LDLR-/-) mice after 3 and 6 weeks (107,000 versus 28,000 microm(2) [P:<0.001] and 121,000 versus 87,000 microm(2) [P:<0.05], respectively) of an atherogenic diet. LDL from the LDLR-/-/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR-/- mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti-oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.

An actinomycete strain No. Q996-17 produced a novel compound, epoxomicin, which exhibited in vivo antitumor activity against B16 melanoma. Structural studies indicated that it is a new member of the epoxy-beta-aminoketone group, and is closely related to eponemycin.

OBJECTIVE: The capacity of the non-nucleoside reverse transcriptase inhibitor efavirenz to induce either liver CYP3A4 or intestinal CYP3A4, or both, as well as intestinal P-glycoprotein, was evaluated in healthy volunteers during and after a 10-day treatment course with two different daily doses. METHODS: Cohorts of 12 healthy subjects were randomized (2:1) to receive either efavirenz or placebo orally for 10 days. The first cohort received 200 mg efavirenz and the second cohort received 400 mg efavirenz daily. Liver CYP3A4 activity was evaluated on 9 different occasions with use of the erythromycin breath test (ERMBT). Intestinal biopsy specimens were obtained before the first dose of efavirenz and on the day after administration of the last dose to measure intestinal CYP3A4 and P-glycoprotein contents by immunoblotting. Efavirenz plasma levels were measured by HPLC, and pharmacokinetic parameters were determined by standard noncompartmental methods. RESULTS: Efavirenz significantly increased the mean ERMBT result in a dose- and time-dependent manner, with a 55% mean induction at 400 mg and a 33% mean induction at 200 mg (P <.01, compared with placebo for each treatment). The efavirenz AUC on day 10 correlated with the magnitude of induction (day 11/day 1 ERMBT ratio) when the two dose groups were combined (r = 0.509; P =.04). In contrast, efavirenz treatment had no detectable effect on intestinal CYP3A4 or P-glycoprotein. CONCLUSIONS: Efavirenz is an inducer of liver CYP3A4 in healthy volunteers, and interpatient differences in magnitude of induction is partly explained by variation in systemic drug exposure. However, efavirenz did not appear to induce intestinal CYP3A4 or intestinal P-glycoprotein. These results suggest that drug interactions caused by induction of CYP3A4 can be liver specific.

Bacillaene, a novel polyene antibiotic, was discovered and isolated from fermentation broths of a strain of Bacillus subtilis. The novel antibiotic has a nominal molecular weight of 580 and an empirical formula of C35H48O7. Bacillaene is active against a broad spectrum of bacteria in agar-plate diffusion assays. Studies in vitro indicate that the antibiotic inhibits prokaryotic protein synthesis but not eukaryotic protein synthesis. Cell survival studies performed with strains of Escherichia coli indicate that the antibiotic is a bacteriostatic agent.

Integrins play an important role in regulating cell adhesion, motility, and activation. In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of β1-integrin (CD29), we used the β1-integrin cytoplasmic domain as bait in the yeast two-hybrid system. Here we report that the cytoplasmic domain of β1-integrin specifically interacts with the cytoskeletal protein filamin. This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of β1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4.5 ∼96-residue tandem repeats of filamin. To verify this interaction in vivo, we showed that filamin specifically coprecipitated with β1 in mammalian cells. We also showed that recombinant filamin chimeric proteins were able to bind to the β1 cytoplasmic domain in vitro. We observed that a subset of single point mutations in the cytoplasmic domain of β1, which had been previously reported to impair its function, disrupt the interaction between β1and filamin. Taken together, these findings suggest that the interaction between β1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function. Integrins play an important role in regulating cell adhesion, motility, and activation. In an effort to identify intracellular proteins expressed by activated T cells that interact with the cytoplasmic domain of β1-integrin (CD29), we used the β1-integrin cytoplasmic domain as bait in the yeast two-hybrid system. Here we report that the cytoplasmic domain of β1-integrin specifically interacts with the cytoskeletal protein filamin. This interaction required all but the most carboxyl-terminal three residues of the cytoplasmic domain of β1, and the carboxyl-terminal 477 residues of filamin containing the terminal 4.5 ∼96-residue tandem repeats of filamin. To verify this interaction in vivo, we showed that filamin specifically coprecipitated with β1 in mammalian cells. We also showed that recombinant filamin chimeric proteins were able to bind to the β1 cytoplasmic domain in vitro. We observed that a subset of single point mutations in the cytoplasmic domain of β1, which had been previously reported to impair its function, disrupt the interaction between β1and filamin. Taken together, these findings suggest that the interaction between β1 and filamin, which in turn can bind actin, provides a mechanism for the interaction of this cell surface receptor with cytoskeletal proteins and that this interaction plays a role in normal receptor function. monoclonal antibody polymerase chain reaction phosphate-buffered saline Specimen Diluent. The integrins are a family of closely related heterodimeric cell surface receptors, which play an important role in mediating cell-extracellular matrix and cell-cell interactions (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8988) Google Scholar). The integrins are composed of an α chain, which is associated with a β chain. To date, 16 α chains and 8 β chains have been characterized at the molecular level. Each α and β chain combination results in a cell surface receptor with a unique ligand specificity. Although both chains are required for ligand binding, it appears that the interaction of integrins with cytoplasmic proteins is predominantly mediated by cytoplasmic residues of the β chain. Typically, integrins are expressed in a low affinity conformation on the surface of resting cells. Cell activation can result in increased numbers of receptors; however, more importantly, cellular activation results in a transient change in receptor affinity that is critical for receptor-ligand interaction. The β1-integrin (CD29) family of receptors is composed of at least 10 members by virtue of the association of β1with at least 10 different α chains, α1–α9, and α v. This family of receptors is involved in mediating interactions between cells and extracellular matrix proteins including laminin, fibronectin, collagen, and vitronectin. We have been particularly interested in studying the α4β1 (VLA-4, or very late antigen-4; CD49d/CD29) receptor. This protein has been shown to have at least two ligands, the extracellular matrix protein fibronectin (2Wayner E.A. Garcia-Pardo A. Humphries M.J. McDonald J.A. Carter W.G. J. Cell Biol. 1989; 109: 1321-1330Crossref PubMed Scopus (635) Google Scholar, 3Mould A.P. Wheldon L.A. Komoriya A. Wayner E.A. Yamada K.M. Humphries M.J. J. Biol. Chem. 1990; 265: 4020-4024Abstract Full Text PDF PubMed Google Scholar, 4Guan J.L. Hynes R.O. Cell. 1990; 60: 53-61Abstract Full Text PDF PubMed Scopus (518) Google Scholar, 5Mould A.P. Humphries M.J. EMBO J. 1991; 10: 4089-4095Crossref PubMed Scopus (165) Google Scholar) and VCAM-1 (INCAM-110, CD106) (6Elices M.J. Osborn L. Takada Y. Crouse C. Luhowskyj S. Hemler M.E. Lobb R.R. Cell. 1990; 60: 577-584Abstract Full Text PDF PubMed Scopus (1526) Google Scholar), a member of the immunoglobulin superfamily expressed on the surface of activated endothelial cells. Both β1 integrins and VCAM-1 have been shown to be critical for normal development. Mice lacking β1 are not viable (7Fassler R. Meyer M. Genes Dev. 1995; 9: 1896-1908Crossref PubMed Scopus (611) Google Scholar), and studies with chimeric mice lacking expression of β1 in blood cells and hematopoietic cells indicate that hematopoietic stem cells lacking β1 can differentiate normally but are unable to populate the fetal liver (8Hirsch E. Iglesias A. Potocnik A.J. Hartmann U. Fassler R. Nature. 1996; 380: 171-175Crossref PubMed Scopus (300) Google Scholar). On the other hand, the majority of mice lacking VCAM-1 have abnormal placental development and die at the embryo stage within 3 days (9Gurtner G.C. Davis V. Li H. McCoy M.J. Sharpe A. Cybulsky M.I. Genes Dev. 1995; 9: 1-14Crossref PubMed Scopus (313) Google Scholar, 10Kwee L. Baldwin H.S. Shen H.M. Stewart C.L. Buck C. Buck C.A. Labow M.A. Development. 1995; 121: 489-503Crossref PubMed Google Scholar); however, a small number of mice lacking VCAM-1 survive and become fertile, and their only observed defect is an elevation in the number of peripheral blood mononuclear leukocytes. In mature individuals the interaction between VLA-4 and its two ligands plays an important role in regulating the recruitment and migration of leukocytes to sites of inflammation. Interactions between VLA-4 and VCAM-1 are in part responsible for mediating the adhesion of leukocytes to activated vascular endothelial cells (11Carlos T.M. Schwartz B.R. Kovach N.L. Yee E. Rosso M. Osborn L. Chi-Rosso G. Newman B. Lobb R. Harlan J.M. Blood. 1990; 76: 965-970Crossref PubMed Google Scholar, 12Schwartz B.R. Wayner E.A. Carlos T.M. Ochs H.D. Harlan J.M. J. Clin. Invest. 1990; 85: 2019-2022Crossref PubMed Scopus (124) Google Scholar), while interactions between VLA-4 and fibronectin play a role in allowing leukocytes to efficiently migrate to the sites of inflammation following leukocyte diapedisis (13Hauzenberger D. Klominek J. Sundqvist K.G. J. Immunol. 1994; 153: 960-971PubMed Google Scholar, 14Molossi S. Elices M. Arrhenius T. Rabinvitch M. J. Cell. Physiol. 1995; 164: 620-633Crossref PubMed Scopus (24) Google Scholar, 15Ratner S. Invasion Metatasis. 1992; 12: 82-100PubMed Google Scholar). The contribution of α4 to the recruitment of leukocytes to sites of inflammation in mature animals has been demonstrated by examining the effects of anti-α4mAbs1 in various models of immune disease. Antibodies directed against α4, VLA-4, and VCAM-1 have been shown to block antigen-induced eosinophil and T cell infiltration into bronchial tissue (16Pretolani M. Ruffie C. Lapa e Silva J.-R. Joseph D. Lobb R.R. Vargaftig B.B. J. Exp. Med. 1994; 180: 795-805Crossref PubMed Scopus (167) Google Scholar, 17Nakajima H. Sano H. Nishimura T. Yoshida S. Iwamoto I. J. Exp. Med. 1994; 179: 1145-1154Crossref PubMed Scopus (287) Google Scholar). Using an experimental autoimmune encephalomyelitis model, anti-α4 antibodies have been shown to prevent the accumulation of lymphocytes in the central nervous system and the development of experimental autoimmune encephalomyelitis (18Yednock T.A. Cannon C. Fritz L.C. Sanchez M., F. Steinman L. Karin N. Nature. 1992; 356: 63-66Crossref PubMed Scopus (1509) Google Scholar). Antibodies to α4 and VCAM-1 have also been shown to delay the onset of diabetes in an adoptive transfer model of insulin-dependent diabetes mellitus and block lymphocyte infiltration of the islets of Langerhans (19Baron J.L. Reich E.-P. Visintin I. Janeway C.A.J. J. Clin. Invest. 1994; 93: 1700-1708Crossref PubMed Scopus (109) Google Scholar). Anti-α4 antibodies also lower leukocyte infiltration at sites of inflammation in an in vivo contact hypersensitivity model (20Ferguson T.A. Kupper T.S. J. Immunol. 1993; 150: 1172-1182PubMed Google Scholar, 21Chisholm P.L. Williams C.A. Lobb R.R. Eur. J. Immunol. 1993; 23: 682-688Crossref PubMed Scopus (96) Google Scholar). These effects may reflect blocking of the interaction between VLA-4 and/or α4/β7-integrins and their respective ligands. In a similar experiment, a soluble VCAM-1-Ig fusion protein was shown to inhibit the onset of insulin-dependent diabetes in an adoptive transfer model and delay the appearance of islet-specific leukocyte infiltration (22Jakubowski A. Ehrenfels B.N. Pepinsky R.B. Burkly L.C. J. Immunol. 1995; 155: 938-946PubMed Google Scholar). We have been interested in the interaction between VLA-4 and VCAM-1 and have examined the role of this interaction in cell adhesion and signaling. Several groups, including ours, have found that VCAM-1 and fibronectin to VLA-4 can lymphocyte cell adhesion and Both the cell that cell adhesion Y. S. Nature. 1990; PubMed Scopus Google Scholar, R. T.A. Full Text Full Text PDF Scopus Google Scholar, A. J. Immunol. 1994; Google Scholar, Y. E. Newman S. J. Exp. Med. 1992; PubMed Scopus Google Scholar, C. R. B. T.A. J. Cell Biol. 1996; PubMed Scopus Google Scholar) and the that lymphocyte adhesion, and migration A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Clin. Invest. 1993; PubMed Scopus Google Scholar, J.A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar) are for the most part mediated by interactions between the cytoplasmic domain of β1 and a number of intracellular proteins (1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8988) Google Scholar, C. L. G. J. S. Nature. 1996; PubMed Scopus Google Scholar, M.A. Cell Dev. Biol. 1995; PubMed Scopus Google Scholar). In this we the of the yeast two-hybrid system to identify an interaction between the cytoskeletal protein filamin and the cytoplasmic domain of This interaction was by examining the of filamin to with β1 in the T cell we that recombinant filamin chimeric proteins were able to bind to the β1 cytoplasmic domain in vitro. 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U. S. A. 1993; PubMed Scopus Google Scholar). The R.R. U. S. A. 1990; PubMed Scopus Google Scholar) as a cell were by of cells in of containing and with Cell were with of the for at were by of of protein on and were with against or filamin J. A. J. J. Immunol. 1992; Google Scholar). were with antibodies against and In an effort to identify cytoplasmic proteins that bind to the cytoplasmic domain of β1, we a the cytoplasmic domain of β1 into the bait This was used in with a activated T cells and in the to for β1 proteins the yeast two-hybrid system as of in the of two that a which specifically bind to the cytoplasmic domain of The were in a with or Both of the with the but not the as the activation not of these that were and the carboxyl-terminal of the cytoskeletal protein filamin We also observed that the cytoplasmic domain of the which is to the cytoplasmic domain of β1, interact with the filamin in the yeast two-hybrid system not studies have shown that filamin is an R. S. Stewart M. J. Cell Biol. 1990; PubMed Scopus Google Scholar). Each of an domain by tandem in which for the of filamin. The carboxyl-terminal of the terminal tandem a that for This is the repeats by an The filamin found to be of with β1 the carboxyl-terminal 4.5 tandem repeats including the We two to verify the interaction. we examined the of a recombinant chimeric containing the carboxyl-terminal of filamin, to bind to a to the shown in the interaction between and the to the β1 cytoplasmic domain was and and was not by the of not proteins to the β1 cytoplasmic domain of to the β1 cytoplasmic domain is shown in B. results were a recombinant filamin chimeric protein not we to β1 with filamin in mammalian cells. Cell the T cell were with a or a against β1, and filamin was by with a against filamin. shown in filamin specifically coprecipitated with β1 filamin was in the We were unable to β1 in This is not the results reported by M.A. J. Immunol. 1995; Google Scholar), were able to filamin with an but were unable to with the protein was in not To the β1 and filamin protein required for the interaction of these two we a of containing different of of these two proteins and examined their to interact the yeast two-hybrid system. of 10 filamin were filamin lacking tandem the The other filamin lacking tandem the of the filamin with β1 We observed a interaction of β1 with the containing the tandem repeats but the and carboxyl-terminal tandem that the domain not of β1 cytoplasmic domain with and carboxyl-terminal filamin was with and the residues of the filamin and were on lacking and were and for as and we a of containing different of the cytoplasmic domain of β1 and examined their to bind filamin the yeast two-hybrid system. We observed with in the cytoplasmic domain of the interaction. The only that was was the of the carboxyl-terminal three residues of β1 and of β1 cytoplasmic domain with filamin. S. was with residues and the residues of and were on lacking and were and for as and number of residues in the cytoplasmic domain of β1 have been shown to be critical for the of integrins Y. J. Cell Biol. 1992; 1321-1330Crossref PubMed Scopus Google Scholar, R.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the molecular for these effects is not We to residues that had been previously shown to be important for β1 were involved in the interaction. of single point mutations in the cytoplasmic domain of β1 were and their to bind to filamin was examined the yeast two-hybrid system. studies predominantly the two found in the cytoplasmic domain of The in both was to or and the in both was to or We also the in the it with and it with the with in the of of the with also the while of the with had and of the in the with or the while of the in the carboxyl-terminal with or had and of the in both with had on the interaction. we observed that the in the with and to the interaction and of β1 cytoplasmic domain point with filamin. S. was with residues and the the of the change in the β1 and were on lacking and were and for as and the interactions between the cytoplasmic domain of the β chain of the integrins with intracellular proteins is critical to of the molecular that function. we that the cytoplasmic domain of β1 can bind to the cytoskeletal protein filamin. The interaction was the as bait in a yeast two-hybrid The interaction between β1 and filamin be demonstrated by studies that showed that filamin specifically coprecipitated with β1 in the T cell the protein was in β1 of a interaction between β1 and filamin. fusion proteins containing the carboxyl-terminal of filamin specifically to a containing the cytoplasmic domain of β1 in vitro. with a of β1 and filamin showed that the interaction between these two proteins in the yeast two-hybrid system required all but the most carboxyl-terminal three of β1and the carboxyl-terminal tandem repeats of filamin that the is these are the required for vivo or these are the required to the interaction the yeast two-hybrid system. The of a interaction between β1 and the carboxyl-terminal filamin lacking the and tandem may reflect a of of the yeast two-hybrid system to very is of to that the has also been shown to bind to filamin D. Newman R.R. J. Cell Biol. PubMed Scopus (109) Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the domain has been to the of filamin the S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). filamin may with proteins that interact at sites on filamin. we that residues in the in the cytoplasmic domain of β1, which had been shown previously to play a role in β1 function, are critical for filamin these the and residues to be the most important for the is with or the interaction between β1 and filamin is while of with but not the interaction. the in the with or but not On the other hand, similar at the had on the of of the in the with the and of a that had also been shown to be important for with β1 to filamin. findings on the interaction between β1 and filamin are with an interaction of filamin with S. Yamada K.M. 1995; PubMed Scopus Google Scholar) showed that with fibronectin, or antibodies to the or β1 of filamin in In a M.A. J. Immunol. 1995; Google Scholar) showed by a that filamin with the cytoplasmic domain of a closely related β chain. These three to the of the cytoplasmic domain of the carboxyl-terminal of the cytoplasmic domain of or a which is a of and residues of These were to and their to filamin These reported that only was of filamin. of the interaction β1 is with this We also observed that of residues the interaction. the that which is in both and β1 and is within the to the interaction is with their that residues in the of the cytoplasmic domain of are required for filamin the the and studies suggest that the interaction between β1 and filamin residues in the carboxyl-terminal of the cytoplasmic domain of This may reflect between the and cytoplasmic of the of between the two it more that the observed may result in of the the is to that the cytoplasmic domain has two at the the as the in studies indicate that the interaction the of with that these not to be as important to the interaction as are to the can an interaction with filamin in the of the of the interaction to the of with in the that is not required for the interaction. Several had previously reported on a of domain and point that different of β1 function. J.L. Hynes R.O. Cell 1990; PubMed Scopus Google Scholar) a of β1 cytoplasmic domain and showed that β1 lacking only the carboxyl-terminal residues the to to while not efficiently to Y. B. A. D. A. J. Cell Biol. 1990; PubMed Scopus Google Scholar) reported that of residues the of β1 its to to We observed that of the carboxyl-terminal three residues of β1 not its to interact with filamin, while of or more carboxyl-terminal residues its interaction with filamin. Y. J. Cell Biol. 1992; 1321-1330Crossref PubMed Scopus Google Scholar) examined the role of residues in the cytoplasmic domain of β1 on to These all residues in the cytoplasmic domain of the residues which found the of β1 to were and the and residues in the of β1 and the and residues in the carboxyl-terminal of We observed that with the of at the residues in the carboxyl-terminal mutations which adhesion also the interaction between β1 and filamin. in a these showed that a more of with not prevent to Y. B. A. D. A. J. Cell Biol. 1990; PubMed Scopus Google Scholar). we found that the interaction the of with R.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) the studies on these point mutations by examining the effects of single point mutations in the cytoplasmic domain of β1 on number of have been shown to bind to integrins and be In the interaction between and β1 has been These studies have that this to a number of different β1 integrins the protein to and a of the at the of These found that mutations at the and residues in the and but not at the carboxyl-terminal and The of mutations at were not examined in this The that β1 mutations which also filamin binding, in with the that filamin actin, may a molecular for the in the observed following least different β1 have been which in the of their cytoplasmic and F. F. L. G. J. Cell Biol. 1993; 121: PubMed Scopus Google F. G. C. F. G. L. 1990; PubMed Scopus Google Scholar) and E. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, Takada Y. M. 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These studies suggest that the two but not play a role in to These studies suggest that the filamin and sites on β1 In an J.M. Schwartz M.A. Biol. Cell. 1995; PubMed Scopus Google Scholar) examined the of various cytoplasmic domain of β1 to with actin, and adhesion in cells. observed that β1 which the carboxyl-terminal with but not with actin, while a lacking the residues of β1, including the in the carboxyl-terminal of β1, was able to with both and that that the but not the in the carboxyl-terminal of β1, is important for filamin binding, and that both and filamin are the results reported by J.M. Schwartz M.A. Biol. Cell. 1995; PubMed Scopus Google Scholar) suggest that in their experimental system the association between β1 and be mediated by filamin. proteins have been yeast two-hybrid that to function. C. L. G. J. S. Nature. 1996; PubMed Scopus Google Scholar) a which interacts with and the β1 cytoplasmic of this protein in cells the of these cells to to extracellular matrix B. L. S. H. B. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) a cytoplasmic protein that a domain and a domain to the yeast and interacts with the cytoplasmic of this results in activation of and of cells to of the domain also while of the domain to of adhesion to T. M. V. Williams M. J. Cell Biol. 1995; PubMed Scopus Google Scholar) a which specifically interacts with the The role of in to be on it be of to the interaction of filamin with plays a role in the of by these cytoplasmic The of the cytoplasmic domain of β1 to bind filamin provides an molecular mechanism for the of the cytoplasmic domain of this to Although studies have been to the residues in the cytoplasmic domain of β1 which are critical for binding, the to suggest that both and filamin bind to similar sites on β1 and a mechanism for is on the of these two interactions on function. The findings in this the for that the role of filamin in β1 function. We for of and for and for in of the for for in of this and

In the course of screening for new antibiotics active against fungi, an actinomycete strain No. PI57-2 that had been isolated from a soil sample collected in Fiji Island was found to produce novel antibiotics, pradimicins A and B1*^. Pradimicin A, the major component showed moderate in vitro activity against a wide variety of fungi and yeasts including clinically important pathogens. More interestingly, it exhibited marked in vivo therapeutic activity against systemic fungal infections caused by Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans strains in mice. Degradation studies revealed that pradimicin A has a unique structure containing the following moieties : D-Alanine, D-xylose, 4 ,6-dideoxy-4-methylamino-D-galactose and a substituted 5,6-dihydrobenzo[a]naphthacenequinone. This communicationpresents production, isolation, chemical and biological properties and structures of pradimicins A and B.

X-linked hyper IgM syndrome (XHIM) is a primary immunodeficiency disorder caused by mutations of the gene encoding CD40 ligand (CD40L). We correlated mutations of the CD40L gene, CD40L expression, and the clinical manifestations observed in XHIM patients from 30 families. The 28 unique mutations identified included 9 missense, 5 nonsense, 9 splice site mutations, and 5 deletions/insertions. In 4 of 9 splice site mutations, normally spliced and mutated mRNA transcripts were simultaneously expressed. RNase protection assay demonstrated that 5 of 17 mutations tested resulted in decreased levels of transcript. The effect of the mutations on CD40L expression by activated peripheral blood mononuclear cells (PBMC) and T-cell lines or clones was assessed using one polyclonal and four monoclonal antibodies and a CD40-Ig fusion protein. In most patients, the binding of at least one antibody but not of CD40-Ig was observed, suggesting nonfunctional CD40L. However, activated PBMC from three patients and activated T-cell lines from two additional patients, each with different genotype, bound CD40-Ig at low intensity, suggesting functional CD40L. Thus, failure of activated PBMC to bind CD40-Ig is not an absolute diagnostic hallmark of XHIM and molecular analysis of the CD40L gene may be required for the correct diagnosis. Patients with genotypes resulting in diminished expression of wild-type CD40L or mutant CD40L that can still bind CD40-Ig appear to have milder clinical consequences.

BMY 28142, a new broad-spectrum semisynthetic cephalosporin, was evaluated in vitro and in vivo in comparison with ceftazidime, cefotaxime, moxalactam, and cefoperazone. The activity of BMY 28142 compared favorably with the activities of the other compounds against both Pseudomonas aeruginosa and Staphylococcus aureus and was somewhat greater against members of the family Enterobacteriaceae. The influence of inoculum size on MICs of BMY 28142 was small for most of the isolates tested, except Enterobacter species. With Enterobacter strains, a marked inoculum effect was found with all of the compounds, and the effect was more pronounced in broth than agar. Still, MICs of BMY 28142 in broth did not exceed 16 micrograms/ml. Of 37 Enterobacteriaceae strains resistant to one or more of the comparison beta-lactams, none were resistant, at a low inoculum size (10(4) CFU), to BMY 28142, compared with 3 for moxalactam, 18 for ceftazidime, 23 for cefotaxime, and 34 for cefoperazone; at an inoculum size of 10(6) CFU, the number of resistant strains was 12, 17, 25, 34, and 37, respectively. Binding to human serum proteins approximated 19%. Recovery of 73% of the drug in mouse urine indicated good bioavailability. The in vitro profile was sustained in vivo by the results obtained with experimental infections in mice. BMY 28142 was as effective as ceftazidime against systemic infection with P. aeruginosa and as effective as cefotaxime against systemic infection with S. aureus. Overall, infections with 18 of 20 strains representing nine genera were responsive to BMY 28142 at doses equivalent to or lower than those of the most effective of the comparison compounds.

A new antibiotic, cispentacin, was isolated from the culture broth of a Bacillus cereus strain, L450-B2. The antibiotic is water-soluble and amphoteric; its structure was determined by spectroscopic analysis and chemical synthesis to be (1R,2S)-2-aminocyclopentane-1-carboxylic acid. Cispentacin demonstrated only weak in vitro activity against certain fungi but strong protection of mice from lethal infection of Candida albicans A9540.

New antibiotic pumilacidins A, B, C, D, E, F and G were isolated from the culture broth of a strain of Bacillus pumilus. They are cyclic acylheptapeptide composed of a beta-hydroxy fatty acid, two L-leucine, two D-leucine, L-glutamic acid, L-aspartic acid and L-isoleucine (or L-valine). Pumilacidin components were inhibitory to herpes simplex virus type 1 and H+, K(+)-ATPase and demonstrated antiulcer activity in rat.

By means of a rat aortic smooth muscle (RASM) cell culture model, the effects of angiotensin II (AII) on early proto-oncogene gene expression, DNA synthesis, and cell proliferation were measured and compared to known mitogens. In 24-h [3H]-thymidine incorporation assays, AII was found to be a weak mitogen when compared to potent mitogens such as fetal bovine serum and platelet-derived growth factor (PDGF). In contrast, when assays were carried out for 48 h, AII induced a significant dose-dependent stimulation of DNA synthesis, which more than doubled at 3 nM AII, and was maximal (five- to eightfold above control) at 100 nM AII. Treatment of cells with the AII type 1 receptor antagonist losartan inhibited the mitogenic effects of AII. AII also stimulated smooth muscle cell proliferation, as indicated by an absolute increase in cell number after AII stimulation of RASM cells for 5 d. AII stimulation of RASM cell growth correlated with the increased expression of specific endogenous growth factors, including transforming growth factor beta 1 (TGF-beta 1) and PDGF A-chain. However, addition of either PDGF- or TGF-beta 1-neutralizing antibodies failed to significantly reduce the delayed mitogenic effects induced by AII. In contrast, we found that AII-stimulated mitogenesis could be inhibited in a dose-dependent manner by the growth factor inhibitor drug suramin. Taken together, our results indicate that enhanced endogenous growth factor expression may represent the direct mechanism by which AII promotes smooth muscle cell growth in some vascular hyperproliferative diseases.

BACKGROUND: Recently we reported the development and optimization of a zebrafish teratogenicity assay using dechorionated AB strain embryos, a promising assay that was 87% concordant in correctly identifying in vivo teratogens and non-teratogens from a set of 31 compounds (Brannen et al., 2010: Birth Defects Res 89:66-77). METHODS: This assay utilizes a zebrafish morphological score system to characterize adverse effects and identify the no-observed-adverse-effect level (NOAEL). RESULTS: This report describes in detail the morphological score system used in the dechorionated zebrafish embryo culture teratogenicity assay. The morphological assessment includes evaluation of most structures and organ systems and grades relative severity of abnormalities. CONCLUSIONS: To this end, the morphological score system provides information of tissue-specific teratogenicity that has been found to have good concordance with structures found affected in vivo and can also be used to rank compounds based on the severity of malformations.