Role of heparan sulfate in entry and exit of Ross River virus glycoprotein-pseudotyped retroviral vectors.

Variants of Ross River virus (RRV) that bind to heparan sulfate (HS) were previously selected by serial passaging in cell culture. To explore the effects of mutations that convey HS utilization, we pseudotyped Moloney murine leukemia virus (MoMLV), with the RRV envelope. We substituted amino-acid residues 216 and 218 on RRV-E2-envelope glycoprotein with basic amino-acid residues, because these mutations confer affinity for HS upon RRV. However, T216R-RRV- and N218R-RRV-pseudotyped viruses possessed lower transduction titers, and we demonstrated that HS-affinity impeded release of pseudotyped virus from producer cells. Addition of heparinase to HS-expressing target cells reduces the transduction efficiency of the T216R-RRV- and N218R-RRV-pseudotyped viruses, whereas no such effect is seen in cells lacking HS. Under appropriate conditions, these T216R-RRV- and N218R-RRV-pseudotyped viruses have enhanced capacities for transducing HS-expressing cells. General principles concerning viral adaptation to the use of attachment factors and design of pseudotyped viral vectors are discussed.

Oligonucleotide antiviral therapeutics: antisense and RNA interference for highly pathogenic RNA viruses.

RNA viruses are a significant source of morbidity and mortality in humans every year. Additionally, the potential use of these viruses in acts of bioterrorism poses a threat to national security. Given the paucity of vaccines or postexposure therapeutics for many highly pathogenic RNA viruses, novel treatments are badly needed. Sequence-based drug design, under development for almost 20 years, is proving effective in animal models and has moved into clinical trials. Important advances in the field include the characterization of RNA interference in mammalian cells and chemical modifications that can dramatically increase the in vivo stability of therapeutic oligonucleotides. Antisense strategies utilize single-stranded DNA oligonucleotides that inhibit protein production by mediating the catalytic degradation of target mRNA, or by binding to sites on mRNA essential for translation. Double-stranded RNA oligonucleotides, known as short-interfering RNAs (siRNAs), also mediate the catalytic degradation of complementary mRNAs. As RNA virus infection is predicated on the delivery, replication, and translation of viral RNA, these pathogens present an obvious target for the rapidly advancing field of sequence-specific therapeutics. Antisense oligonucleotides or siRNAs can be designed to target the viral RNA genome or viral transcripts. This article reviews current knowledge on therapeutic applications of antisense and RNA interference for highly pathogenic RNA viral infections.

Catecholamines mediate stress-induced increases in peripheral and central inflammatory cytokines.

Proinflammatory cytokines act at receptors in the CNS to alter physiological and behavioral responses. Exposure to stressors increases both peripheral and central proinflammatory cytokines, yet the mechanism(s) of induction remain unknown. Experiments here examined the role of catecholamines in the in vivo induction of proinflammatory cytokines following tailshock stress. Rats were pretreated i.p. with 2.0 mg/kg prazosin (alpha1-adrenoceptor antagonist), 10.0 mg/kg propranolol (beta-adrenoceptor antagonist), or 5.0 mg/kg labetalol (alpha1- and beta-adrenoceptor antagonist) 30 min prior to tailshock exposure and plasma interleukin-1beta (IL-1beta) and IL-6, along with tissue interleukin-1beta from the hypothalamus, hippocampus, and pituitary were measured immediately following stressor termination. Prazosin attenuated stress-induced plasma IL-1beta and IL-6, but had no effect on tissue IL-1beta levels, while propranolol attenuated plasma IL-6 and blocked tissue IL-1beta elevation, and labetalol, which cannot cross the blood-brain barrier, attenuated plasma IL-1beta and IL-6, blocked pituitary IL-1beta, but had no effect on central tissue IL-1beta levels. Furthermore, administration of 50.0 mg/kg N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride, a neurotoxin that lesions neural projections from the locus coeruleus, prevented stress-induced elevation in hippocampal IL-1beta, a region highly innervated by the locus coeruleus, but had no effect on hypothalamic IL-1beta, a region that receives few locus coeruleus projections. Finally, i.p. injection of 5.0 mg/kg isoproterenol (beta-adrenoceptor agonist) was sufficient to induce circulating IL-1 and IL-6, and tissue IL-1beta. These data suggest catecholamines play an important role in the induction of stress-induced proinflammatory cytokines and that beta-adrenoceptors are critical for tissue IL-1beta induction, while both alpha- and beta-adrenoceptors contribute to the induction of plasma cytokines.

Splenic norepinephrine depletion following acute stress suppresses in vivo antibody response.

Exposure to an intense acute stressor immediately following immunization leads to a reduction in anti-KLH IgM, IgG, and IgG2a, but not IgG1. Stress also depletes splenic norepinephrine (NE) content. Immunization during pharmacological (alpha-methyl-p-tyrosine) or stress-induced splenic NE depletion results in antibody suppression similar to that found in rats immunized prior to stressor exposure. Prevention of splenic NE depletion during stress by tyrosine, but not pharmacological elevation (mirtazapine) of NE, resulted in normal antibody responses. These data support the hypothesis that splenic NE depletion is necessary and sufficient for stress-induced suppression of antibody to a T-cell dependent antigen.

Transplant Friends: an interactive education program for patients awaiting kidney transplantation.

BACKGROUND: Organ transplantation is the preferred treatment for end-stage renal disease. Renal transplant recipients are surviving longer with a better quality of life. Although many hospitals have transplant education programs in place, transplant patients indicate that there is a need for additional information. Transplant Friends is a program designed to meet these needs. PATIENTS AND METHODS: At the University of Alberta, 128 patients attended the Transplant Friends program between September 2002 and February 2003. Each patient completed an evaluation form consisting of 15 questions designed to evaluate patient's satisfaction regarding session content, ease of scheduling, and the sessions facilitators. Responses were recorded using 5-point Likert scales. RESULTS: All 128 participants completed the questionnaires. The predominantly male (59.1%) and Caucasian (91.6%) population had a median age of 49.1 years. Of the 128 patients, 110 patients (86%) felt that the content of the program met or exceeded their expectations; 120 patients (94%) felt the program facilitators met or exceeded their expectations; and 113 patients (88%) evaluated the scheduling favorably. CONCLUSION: Patients require complete information prior to renal transplantation to make an informed decision about whether to proceed with transplant as well as to enhance the overall transplant experience. Patients evaluated the Transplant Friends program as successfully meeting these needs through a comprehensive interactive teaching program. We recommend that institutions performing renal transplants incorporate an educational program such as Transplant Friends during the workup process of this unique patient population.

A novel family of hydroxamate-based acylating inhibitors of cyclooxygenase.

Aspirin irreversibly inhibits cyclooxygenase (COX) by acetylating a serine residue in the active site. We synthesized a series of novel acylating agents based on our previously reported acetylating compound, O-acetylsalicylhydroxamic acid. One of these, triacetylsalicylhydroxamic acid (TriAcSHA) was more effective than aspirin and O-acetylsalicylhydroxamic acid in inactivating both COX-1 and COX-2. Preincubation of COX-1 with inhibitor for 5 min yielded IC(50) values of 18 microM for TriAcSHA and 60 microM for acetylsalicylic acid. Inhibition was time-dependent, with complete inhibition within 10 min at a concentration of 50 microM. As with aspirin, mutation of the serine 530 of COX-1 to alanine abolished the activity of the TriAcSHA. Mutation of the alanine 119 to a glutamine markedly reduced the sensitivity to TriAcSHA, suggesting that this residue was necessary for the interaction with the enzyme. TriAcSHA was also more effective than aspirin as an inhibitor of platelet aggregation induced by arachidonic acid. The diacetylated phenylhydroxamates N-methyl-O,O-diacetylsalicylhydroxamic acid, N,O-diacetylbenzohydroxamic acid, and 2-methyl-O,N-diacetylbenzohydroxamic acid showed reduced or absent activity against COX-1. In addition, we synthesized a series of triacylsalicylhydroxamic acids with progressively longer acyl groups (three to six carbons). All of the compounds inhibited COX-1 and demonstrated progressively greater COX-1 selectivity with increasing number of carbons. Hence, salicylhydroxamic acid provides a versatile backbone for the generation of a family of acylating inhibitors of cyclooxygenase.

In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins.

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 x 10(8) TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.

O-acetylsalicylhydroxamic acid, a novel acetylating inhibitor of prostaglandin H2 synthase: structural and functional characterization of enzyme-inhibitor interactions.

Aspirin is unique among clinically used nonsteroidal antiinflammatory drugs in that it irreversibly inactivates prostaglandin (PG) H2 synthase (PGHS) via acetylation of an active-site serine residue. We report the synthesis and characterization of a novel acetylating agent, O-acetylsalicylhydroxamic acid (AcSHA), which inhibits PGE2 synthesis in vivo and blocks the cyclooxygenase activity of PGHS in vitro. AcSHA requires the presence of the active-site residue Ser-529 to be active against human PGHS-1; the S529A mutant is resistant to inactivation by the inhibitor. Analysis of PGHS inactivation by AcSHA, coupled with the X-ray crystal structure of the complex of ovine PGHS-1 with AcSHA, confirms that the inhibitor elicits its effects via acetylation of Ser-529 in the cyclooxygenase active site. The crystal structure reveals an intact inhibitor molecule bound in the enzyme's cyclooxygenase active-site channel, hydrogen bonding with Arg-119 of the enzyme. The structure-activity profile of AcSHA can be rationalized in terms of the crystal structure of the enzyme-ligand complex. AcSHA may prove useful as a lead compound to facilitate the development of new acetylating inhibitors.

Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations.

Nonsteroidal antiinflammatory drugs (NSAIDs) block prostanoid biosynthesis by inhibiting prostaglandin H(2) synthase (EC 1.14.99.1). NSAIDs are either rapidly reversible competitive inhibitors or slow tight-binding inhibitors of this enzyme. These different modes of inhibition correlate with clinically important differences in isoform selectivity. Hypotheses have been advanced to explain the different inhibition kinetics, but no structural data have been available to test them. We present here crystal structures of prostaglandin H(2) synthase-1 in complex with the inhibitors ibuprofen, methyl flurbiprofen, flurbiprofen, and alclofenac at resolutions ranging from 2.6 to 2.75 A. These structures allow direct comparison of enzyme complexes with reversible competitive inhibitors (ibuprofen and methyl flurbiprofen) and slow tight-binding inhibitors (alclofenac and flurbiprofen). The four inhibitors bind to the same site and adopt similar conformations. In all four complexes, the enzyme structure is essentially unchanged, exhibiting only minimal differences in the inhibitor binding site. These results argue strongly against hypotheses that explain the difference between slow tight-binding and fast reversible competitive inhibition by invoking global conformational differences or different inhibitor binding sites. Instead, they suggest that the different apparent modes of NSAID binding may result from differences in the speed and efficiency with which inhibitors can perturb the hydrogen bonding network around Arg-120 and Tyr-355.

Ross River virus glycoprotein-pseudotyped retroviruses and stable cell lines for their production.

Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.

Plasmid transfection and retroviral transduction of porcine muscle cells for cell-mediated gene transfer.

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not been developed for pigs. The objectives of the present study were to determine methods for transient and stable incorporation of reporter genes into porcine muscle cells and to investigate their use for cell-mediated gene transfer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-specific monoclonal antibodies, myotube fusion assays, and the presence of muscle-specific markers (MyoD and desmin). Four commercial cationic liposomes (lipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in the greatest (P < .01) transient luciferase activity for both cell types. Electroporation of cells for transient transfection resulted in less luciferase activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the neomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electroporation resulted in efficiencies that were significantly lower than established with cationic liposomes. Porcine cells were transduced with GFP using vesicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These results show that cationic liposomes are superior to electroporation for transfection, but retroviral transduction produced stable reporter gene expression in > 80% of porcine muscle cells. Transduced GFP-positive cells were separated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation resulted in limited GFP expression within muscle, whereas myoblast implantation resulted in GFP within muscle fibers. This suggests that cell-mediated gene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.

Crystallization and preliminary X-ray crystallographic analysis of the electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein from the rumen bacterium Megasphaera elsdenii is a heterodimer (M(r) = 75 kDa) containing FAD as cofactor and functioning solely to mediate electron transfer between the prosthetic groups of other proteins. The enzyme was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as precipitant. The crystals obtained belong to the space group P2(1)2(1)2(1) with unit-cell dimensions of a = 58.75, b = 61.77 and c = 122.27 A. Interestingly the crystals exhibit a low solvent content. Crystals diffracted to beyond 2.5 A using synchrotron radiation.

Swelling of Müller cells induced by AP3 and glutamate transport substrates in rat retina.

Previous studies have shown that a single systemic injection of 2-amino-3-phosphonopropionate (AP3), an agonist/antagonist at metabotropic glutamate receptors, produces marked swelling of rodent Müller cells. To investigate the effects of AP3 on Müller cells, we used in vitro retinal segments prepared from 30 day old rats. Incubation with AP3 for 1 h or more caused severe swelling of Müller cells with the appearance of mitotic cellular profiles in the outer nuclear layer. The Müller cell swelling was mimicked by substrates for glutamate transporters, suggesting that AP3 may produce its effects via transport into glial cells. To determine whether AP3 is a substrate for glutamate transporters, we studied cultured rat hippocampal astrocytes using whole-cell patch clamp recordings. In hippocampal astrocytes, AP3 activated currents via an Na(+)-dependent glutamate transporter. Consistent with this, substitution of extracellular sodium with choline blocked Müller cell swelling in the rat retina. These results indicate that the acute glial swelling produced by AP3 results primarily from a fluid shift that accompanies the transport of AP3 and sodium into Müller cells.

Age dependent sensitivity of the rat retina to the excitotoxic action of N-methyl-D-aspartate.

We have found that the rat retina can be isolated atraumatically and incubated ex vivo for up to 24 h without showing signs of histological deterioration, and that retinas from adult or aged rats can be isolated as successfully as those from immature rats. In the present study we used this preparation to show that rat retinal neurones at postnatal day zero (PND 0) are relatively insensitive to the excitotoxic action of the glutamate agonist, N-methyl-D-aspartate (NMDA), then gradually show increasing sensitivity that peaks at about PND 9 and declines from PND 15-30 after which it remains at a low level up to the last time point studied (10 months of age). This is consistent with other developmental NMDA receptor data and underscores the need for caution in using immature in vitro central; nervous system (CNS) tissue preparations as a basis for interpreting the role of NMDA receptors in adult neurological diseases.

Transient photopenia of the femoral head following arthrography.

In cases of suspected sepsis of the hip, a negative arthrocentesis may precede bone scintigraphy. Routine instillation of contrast material and air for localization and characterization of the hip joint was observed to produce a transient photopenic femoral head on bone scintigraphy. The photopenia, related to venous tamponade created by the temporarily increased intra-articular pressure of the arthrogram, occurs when the isotopic study is performed within a half hour of the arthrocentesis. The cases of four children suspected of having septic arthritis and/or osteomyelitis and ranging in age from 2 to 19 years are presented to illustrate the temporal relationship of reversible femoral head photopenia with sequential arthrocentesis and bone scintigraphy.

Scintigraphic differentiation of congenital soft-tissue extremity enlargement with Tc-99m DTPA.

Radionuclide imaging of benign soft-tissue tumors sometimes associated with extremity enlargement (7 patients) and/or osteodysplasia (6 patients) has demonstrated, in a total of 18 patients, several differentiating patterns of accumulation of Technetium-99m diethylene triamine pentaacetic acid (Tc-99m DTPA). Early imaging (within 15 min) as well as later imaging (one-half hour to 3 hours following the intravenous injection of the radiopharmaceutical) has shown that fatty tumors (lipomas, lipoblastomas, fibrofatty tissue) do not concentrate the isotope. Neurofibromas display gradual intensification of their radioactive content, while hemangiomas differ in their scintigraphic pattern depending on their histologic composition. Purely capillary hemangiomas have transient early intense activity while purely cavernous hemangiomas display no early activity but are well visualized on delayed scintigraphic images. Mixed hemangiomas display combined imaging characteristics of both capillary and cavernous types with the predominant pattern dependent upon the predominant histology. Aggressive fibromatosis exhibited an early fleeting display of intense radioactivity.