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Biomedical subjects

R M Cook

Publications and source records attributed to R M Cook.

At least 19 recordsLinked to original sources
PubMed2002

Preclinical efficacy and safety of pascolizumab (SB 240683): a humanized anti-interleukin-4 antibody with therapeutic potential in asthma.

The type 2 helper T cell (T(H)2) cytokine interleukin (IL)-4 is thought to play a central role in the early stages of asthma. In an effort to develop an antibody treatment for asthma that neutralizes the effects of IL-4, a murine monoclonal antibody, 3B9, was generated with specificity for human IL-4. In vitro studies demonstrated that 3B9 inhibited IL-4-dependent events including IL-5 synthesis, (T(H)2) cell activation and up-regulation of immunoglobulin E expression. 3B9 was then humanized (pascolizumab, SB 240683) to reduce immunogenicity in humans. SB 240683 demonstrated species specificity for both monkey and human IL-4 with no reactivity to mouse, rat, cow, goat or horse IL-4. Pascolizumab inhibited the response of human and monkey T cells to monkey IL-4 and effectively neutralized IL-4 bioactivity when tested against several IL-4-responsive human cell lines. Affinity studies demonstrated rapid IL-4 binding by pascolizumab with a slow dissociation rate. In vivo pharmacokinetic and chronic safety testing in cynomolgus monkeys demonstrated that pascolizumab was well tolerated, and no adverse clinical responses occurred after up to 9 months of treatment. Three monkeys developed an anti-idiotypic response that resulted in rapid pascolizumab clearance. However, in the chronic dosing study the antibody response was transient and not associated with clinical events. In conclusion, pascolizumab is a humanized anti-IL-4 monoclonal antibody that can inhibit upstream and downstream events associated with asthma, including (T(H)2) cell activation and immunoglobulin E production. Clinical trials are under way to test the clinical efficacy of pascolizumab for asthma.

Animals↗
PubMed2001

Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys.

BACKGROUND: Allergic respiratory diseases are characterized by large numbers of eosinophils and their reactive products in airways and blood; these are believed to be involved in progressive airway damage and remodeling. IL-5 is the principal cytokine for eosinophil maturation, differentiation, and survival. Mepolizumab (SB-240563), a humanized monoclonal antibody (mAb) specific for human IL-5, is currently in clinical trials for treatment of asthma. OBJECTIVE: The purpose of this study was to characterize the pharmacologic activity and long-term safety profile of an anti--human IL-5 mAb to support clinical trials in asthmatic patients. METHODS: Naive and Ascaris suum -sensitive cynomolgus monkeys received various dose levels of mepolizumab and were monitored for acute and chronic pharmacologic and toxic responses. RESULTS: To support preclinical safety assessment, cynomolgus monkey IL-5 was cloned, expressed, and characterized. Although monkey IL-5 differs from human IL-5 by 2 amino acids (Ala27Gly and Asn40His), mepolizumab has comparable inhibitory activity against both monkey IL-5 and human IL-5. In A suum--sensitive monkeys, single doses of mepolizumab significantly reduced blood eosinophilia, eosinophil migration into lung airways, and levels of RANTES and IL-6 in lungs for 6 weeks. However, mepolizumab did not affect acute bronchoconstrictive responses to inhaled A suum. In an IL-2--induced eosinophilia model (up to 50% blood eosinophilia), 0.5 mg/kg mepolizumab blocked eosinophilia by >80%. Single-dose and chronic (6 monthly doses) intravenous and subcutaneous toxicity studies in naive monkeys found no target organ toxicity or immunotoxicity up to 300 mg/kg. Monkeys did not generate anti-human IgG antibodies. Monthly mepolizumab doses greater than 5 mg/kg caused an 80% to 100% decrease in blood and bronchoalveolar lavage eosinophils lasting 2 months after dosing, and there was no effect on eosinophil precursors in bone marrow after 6 months of treatment. Eosinophil decreases correlated with mepolizumab plasma concentrations (half-life = 13 days). CONCLUSION: These studies demonstrate that chronic antagonism of IL-5 by mepolizumab in monkeys is safe and has the potential, through long-term reductions in circulating and tissue-resident eosinophils, to be beneficial therapy for chronic inflammatory respiratory diseases.

Animals↗
PubMed2000

A tetramethyl rhodamine (Tamra) phosphoramidite facilitates solid-phase-supported synthesis of 5'-Tamra DNA.

5-Carboxy Tamra 1 was conjugated to 4-hydroxypiperidine with BOP and N-methylmorpholine, and the resulting 5-(N-pipyridyl-4-hydroxy)-Tamra carboxamide 2 was treated with 2-cyanoethyl tetraisopropylphosphorodiamidite to give 5-[N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphoramidite)]-Tamra carboxamide 3. Solutions of 3 were coupled onto the 5'-hydroxyl of solid-phase-supported DNA fragments with standard amidite coupling techniques. Cleavage and deprotection with aqueous tert-butylamine cocktail gave 5-Tamra-functionalized DNA as well as an additional compound without the Tamra chromophore. A mass spectrum of this product showed the incorporation of tert-butylamine. The extra product was completely suppressed by including a 5 min acetylation step after coupling. A model study of 3 coupled onto thymidine-functionalized CPG showed similar results. NMR and mass spectra of cleaved products confirmed the addition of tert-butylamine to the minor product. Coupling a Tamra active ester onto T CPG which was previously coupled with N-(4-methoxytrityl)piperidyl-4-O-(2-cyanoethyl diisopropylphosphoramidite) 4 produced the same major Tamra-bearing product, which coeluted on reverse phase HPLC with the major product generated with 3.

Chromatography, High Pressure Liquid↗
PubMed2000

Inhibition of CD23 processing correlates with inhibition of IL-4-stimulated IgE production in human PBL and hu-PBL-reconstituted SCID mice.

BACKGROUND: CD23, the low affinity serum immunoglobulin E (IgE) receptor, is upregulated on B cells following interleukin (IL)-4 stimulation and is concomitantly cleaved to generate soluble CD23 (sCD23) fragments with cytokine-like activity. OBJECTIVE: Compounds that selectively inhibit the proteolytic release of CD23 to generate sCD23 were assessed for their ability to inhibit IgE production in order to evaluate the contribution of sCD23 in the production of human IgE as well as the ability of such compounds to block IgE production. METHODS: IgE production was measured in IL-4-stimulated human peripheral blood lymphocytes (PBL) and PBL-reconstituted SCID mice in the presence of a broad-spectrum matrix metalloprotease (MMP) inhibitor, a compound selective for inhibition of CD23 processing over MMPs and an anti-CD23 mAb, MHM6. RESULTS: The two compounds were equipotent in inhibiting IgE production without inhibition of IgG production by IL-4/anti-CD40-stimulated PBL. Soluble CD23 release was also shown to precede IgE accumulation in the cell-free medium. Addition of compound at later times other than day 0 in the 14 day assay resulted in progressively less inhibition of both IgE and sCD23, and exactly paralleled the effect of an anti-CD23 mAb, MHM6 on IgE levels. Both compounds also inhibited the release of CD23 from human RPMI 8866 cells adoptively transferred i. p. to mice. Doses required for inhibition of CD23 correlated well with the doses required for inhibition of IgE production in IL-4-challenged hu-PBL-SCID mice. IgE was selectively inhibited over total IgG in the SCID mice as well. CONCLUSIONS: Inhibition of CD23 processing alone is sufficient to inhibit IL-4-stimulated IgE production both in vitro and in vivo.

Animals↗
PubMed2000

Schistosoma mansoni phosphoenolpyruvate carboxykinase, a novel egg antigen: immunological properties of the recombinant protein and identification of a T-cell epitope.

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4(+) Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4(+) Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4(+) Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4(+) Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.

Amino Acid Sequence↗
PubMed1999

A phosphate bound universal linker for DNA synthesis.

A uridine-based linker immobilized onto polystyrene beads at the 5' terminus via a phosphodiester group and then used as a universal DNA synthesis support gives post synthesis DNA cleavage in 8 hrs or less without alkali metal salts. DNA produced with the new support was analyzed by HPLC, MALDI mass spectroscopy and PAGE. Each analysis showed DNA of equivalent quality to that produced with standard CPG supports, without contaminating materials resulting from linker or support backbone decomposition.

Chromatography, High Pressure Liquid↗
PubMed1998

International oral and maxillofacial surgery: the medical/dental interface.

Oral and maxillofacial surgery is a unique specialty based in dentistry but requiring extensive surgical training. The vast proliferation of medical and dental knowledge has resulted in extreme pressure on educational processes in both professions and, as a result, current medical graduates have little knowledge of dentistry and dental education has suffered with a reduction in teaching in medical and surgical disciplines. The specialty of oral and maxillofacial surgery has become the referral base for a wide variety of the surgical and pathological problems in the maxillofacial region. The specialty, therefore, finds itself in a position where its expertise in specialty procedures is frequently poorly understood by both medical and dental colleagues. There is a need for strong international cooperation in order to maintain a viable monospecialty without fragmentation. This must include coordinated training programmes placed on the now widely accepted guidelines for training and education adopted by the International Association of Oral and Maxillofacial Surgeons.

Education, Dental↗
PubMed1998

Mutational inactivation of aminoacylase-I in a small cell lung cancer cell line.

Small cell lung cancer (SCLC) cell lines and tumors invariably exhibit loss of heterozygosity (LOH) or, in rare cases, homozygous deletions involving part or all of chromosome arm 3p, suggesting the presence of 1 or more tumor-suppressor genes in this region. The gene encoding aminoacylase-I (ACYl) is localized on chromosome segment 3p21.1. ACYl enzymatic activity, protein, and mRNA have been demonstrated to be expressed at either undetectable or very low levels in a group of SCLC cell lines and tumors. The demonstration of mutational inactivation of ACYl would support the hypothesis that ACYl inactivation in SCLC confers a selective growth advantage. One of four SCLC cell lines with undetectable Acyl enzymatic activity and protein exhibited a compound mutation: nonconservative missense point mutations at codons 195 and 254. No wildtype sequence transcripts were identified in the cell line. Although nonmutational mechanisms for low or undetectable ACYl enzymatic activity, protein, and mRNA expression are most frequently operant in SCLC, the demonstration of a mutation supports selection for ACYl inactivation. Analysis of normal liver and a liver metastasis from the same patient from whom the NCI-H711 cell line was derived demonstrated that the mutation was neither germline nor an early event in the development of SCLC. It is of interest that several genes involved in the regulation of intracellular protein degradation are encoded by chromosome band 3p21 and display unusual expression in SCLC. The presence of other loci involved in protein degradation on chromosome band 3p21 and their aberrant expression in SCLC suggest that a variety of mechanisms involved in the normal degradation of intracellular proteins may be perturbed in this neoplasm.

Amidohydrolases↗
PubMed1997

IgE secretion is attenuated by an inhibitor of proteolytic processing of CD23 (Fc epsilonRII).

CD23, the low-affinity IgE receptor, is up-regulated on interleukin (IL)-4-stimulated B cells and monocytes, with a concomitant increase in the release of soluble fragments of CD23 (sCD23) into the medium by proteolytic processing of the surface-bound intact CD23. The effect of inhibition of the processing of CD23 on IgE production in human and mouse cells and in a mouse model in vivo was evaluated. CD23 processing to sCD23 from RPMI 8866 (a human Epstein-Barr virus-transformed B cell line) cell membranes was inhibited by a broad-spectrum matrix-metalloprotease inhibitor, batimastat, with an IC50 of 0.15 microM. Batimastat also inhibited CD23 processing in whole RPMI 8866 cells as well as in IL-4-stimulated purified human monocytes with similar IC50. Batimastat inhibited IgE production from IL-4/anti-CD40-stimulated human tonsil B cells as well as mouse splenic B cells in a manner consistent with inhibition of CD23 processing. Release of soluble fragments of CD23 in the cell supernatants of tonsil B cells was inhibited over the concentration range of 1-10 microM batimastat and intact cell surface CD23 was increased on mouse splenic B cells in the presence of these concentrations of batimastat. IgE production of IL-4-stimulated human peripheral blood mononuclear cells was also blocked by 1-10 microM batimastat, again with comparable inhibition of sCD23 release over the same concentration range. Finally, in a mouse model of IgE production, batimastat inhibited IgE production in response to ovalbumin challenge as determined by serum IgE levels. Taken together, the data support a role of CD23 in IgE production and point to CD23 processing to sCD23 as a therapeutically relevant control point in the regulation of IgE synthesis.

Animals↗
PubMed1997

Can feedback be individualised, useful, and economical?

An important, but often neglected, part of any research or audit exercise is the reporting back to participants of the results of that exercise. When feedback is made, it is often of a general, aggregated nature. Considerations of cost and psychological factors usually preclude feedback to individuals. As part of a larger study we have developed a prototype mechanism for providing such individual feedback. This was done by writing a computer program which automatically generated the report, using rules on how to interpret different patterns of responses to a questionnaire. Previous qualitative evaluation had shown a positive response from participating nurses. The current small-scale study reports a more formal evaluation. Participants who received reports on the degree to which their ward was practising primary nursing overwhelmingly found the reports readable, informative, encouraging, accurate and useful.

Communication↗
PubMed1997

Safety of a single aerosol administration of escalating doses of the cationic lipid GL-67/DOPE/DMPE-PEG5000 formulation to the lungs of normal volunteers.

Several groups are assessing the use of cationic lipids for respiratory gene therapy. To date no human data are available regarding the safety of intra-pulmonary cationic lipid delivery. In preparation for a trial of pulmonary delivery of the CFTR gene, we have assessed the safety of nebulised lipid GL-67/DOPE/DMPE-PEG5000 (GL-67A), the cationic lipid formulation to be used in this study. Fifteen healthy volunteers were given incremental doses of GL-67A via a Pari LC Jet nebuliser; three volunteers in each of five dosing cohorts with a week interval between cohorts. Markers of safety included clinical assessment, measurement of lung function, chest CT scan, serological testing and analysis of induced sputum. Measurements were taken before administration and at intervals up to 21 days thereafter. No adverse clinical events were seen or any statistically significant changes in spirometry or gas transfer. There were no clinically significant changes in any of the blood parameters and no CT changes were seen. Comparisons of the cellular subpopulations (neutrophils, eosinophils, lymphocytes and macrophages) in induced sputum showed no significant alterations following administration of the GL-67A. This study suggests that a single application of aerosol formulation of GL-67A does not result in clinically detectable changes when given by nebulisation into the lungs of normal volunteers and provides an indication of a lipid dose tolerated in man.

Aerosols↗
PubMed1996

A new universal linker for solid phase DNA synthesis.

A method is described as an alternative to the use of nucleoside pre-functionalized supports for DNA synthesis. The procedure should allow the generation of 3'-OH terminal moieties of any natural or modified DNA fragment using a single derivatized solid support material. The method utilizes 1-O-(4,4' dimethoxytrityl)-2- O-succinoyl-3-N-allyloxycarbonylpropane immobilized on amino-propyl CPG followed by subsequent coupling of unit phosphoramidites. Work up is accomplished by removal of the 3-N-allyloxycarbonyl group [Pd(0) at 50 degrees C for 15 min] followed by cleavage under very mild conditions (aqueous TEAA/NH3 buffer pH 10, room temperature) to release the desired product. The mechanism is believed to involve nucleophilic attack of the linker-derived amino group on the 3'-phosphate triester, followed by elimination of the desired product. DNA synthesis with the new support and with classical nucleotide synthesis supports have been performed, and the products shown to be identical. Further proof of product integrity was given by MALDI mass spectral studies and the efficacy of DNA primers made with the new support in PCR amplification.

Allyl Compounds↗
PubMed1995

The pyruvate dehydrogenase complex of Saccharomyces cerevisiae is regulated by phosphorylation.

Mitochondria were isolated from Saccharomyces cerevisiae grown on different carbon sources prior to incubation with [gamma-32P]ATP. A major 46,000-M(r) phosphoprotein, corresponding in M(r) value to the E1 alpha subunit of the yeast pyruvate dehydrogenase complex (PDC), was detected only in mitochondria isolated from cells grown on a fermentable carbon source such as galactose. Immunoprecipitation with subunit-specific antiserum to the E1 component of mammalian or yeast PDC confirmed the identity of this polypeptide. PDC activity in isolated yeast mitochondria could be inactivated in an ATP-dependent fashion and reactivated in the presence of Ca2+ ions.

Adenosine Triphosphate↗
PubMed1994

Activation of human blood lymphocytes by house dust mite protein and Mycobacterium tuberculosis purified protein derivative: effects of interferon-gamma, interleukin-4, dexamethasone and cyclosporin A.

Peripheral blood mononuclear cells (PBMC) from nonatopic donors sensitive to Mycobacterium tuberculosis purified protein derivative (PPD) and from atopic donors sensitive to PPD and house dust mite antigen (HDM), were stimulated in vitro to proliferate in response to exogenous IL-2. In the presence of exogenous IFN-gamma, the response of cells from atopic donors to PPD was either unaffected or slightly enhanced, whereas the response to HDM was inhibited in a dose-dependent manner. The response of cells from nonatopic donors to PPD remained unchanged or was only slightly inhibited and this was not dose-dependent. Exogenous IL-4 did not reverse the inhibitory effects of IFN-gamma and a neutralizing antibody to IL-4 did not inhibit the proliferative response of cells to HDM and IL-2. Dexamethasone inhibited the IL-2-mediated response of cells from atopic donors stimulated with HDM, whereas the IL-2-mediated response of cells induced by PPD was either unchanged or stimulated by dexamethasone. Cyclosporin A inhibited the response of cells to both HDM and PPD. These results suggest that IFN-gamma can exert a selective effect on the response of T lymphocytes to different antigens and that it is possible to identify compounds able to regulate this activity.

Animals↗
PubMed1993

Human aminoacylase-1. Cloning, sequence, and expression analysis of a chromosome 3p21 gene inactivated in small cell lung cancer.

Human aminoacylase-1 (N-acyl-L-amino-acid amidohydrolase, EC 3.5.1.14; ACY1) is a homodimeric zinc-binding enzyme that catalyzes the hydrolysis of N alpha-acylated amino acids. ACY1 has been assigned to chromosome 3p21.1, a region reduced to homozygosity in small cell lung cancer (SCLC), and has been reported to exhibit reduced or absent expression in SCLC cell lines and tumors. Two human cDNA libraries and one human genomic DNA library were screened with a previously isolated partial ACY1 cDNA to isolate a full-length transcript. Sequence analysis of clones from each of these libraries resulted in an ACY1 cDNA of 1438 base pairs with an open reading frame of 1224-base pairs coding for a putative protein of 408 amino acids with a predicted molecular mass of 45,882 Da. Sequence analysis revealed no homologies to previously reported cDNA or protein sequences and establishes ACY1 as the first member of a new family of zinc-binding enzymes to be so characterized. The subcellular location of ACY1 has been established as cytosolic by flow cytometry. Southern and northern analyses of ACY1 in SCLC cell lines failed to demonstrate any gross abnormalities of the ACY1 structural gene or instances of absent or aberrantly sized mRNA, respectively.

Amidohydrolases↗