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Biomedical subjects

A P Lewis

Publications and source records attributed to A P Lewis.

At least 19 recordsLinked to original sources
PubMed2004

Influenza circulating strains in Argentina exhibit differential induction of cytotoxicity and caspase-3 in vitro.

BACKGROUND: Human influenza infections are a significant cause of morbidity worldwide. Though damage to the respiratory epithelium and has been related to apoptosis, which occurs subsequent to influenza virus infection, little information is available regarding cell cytotoxicity of human strains. OBJECTIVE: To study cytotoxicity performed in vitro by various circulating strains in Argentina. The study sample consisted of three vaccine strains (H1N1, H3N2, and B) administered during 1999-2000 in South America and three strains isolated from clinical samples, one, NAC (H1N1) obtained from an adult inpatient with human pneumonia; and the other two (T) and (T2) (H3N2) with influenza syndrome. Viral antigen was detected by an immunofluorescence test, conducted prior to viral isolation in MDCK cells. Strains were subtyped by the hemmaglutination inhibition test. Cytotoxic properties were determined by lactate dehydrogenase reaction (LDH), crystal violet staining and Hoechst staining. Caspase-3 activity, morphological changes of apoptosis, and viral yields were measured in MDCK infected cells. RESULTS AND CONCLUSIONS: Cells infected by each of the strains exhibited apoptosis morphology by Hoechst staining and caspase-3 activity was high for both H1N1 strains. Further, high levels of LDH activity were detected for NAC and H3N2 strains tested, indicating the possible role of different viral proteins or functions on cell cytotoxicity. The NAC strain, isolated from human pneumonia and antigenically related to A/New Caledonia /20/99 (H1N1), was the highest cytotoxic strain and an excellent inducer of caspase-3 activity. In turn, no parameter was related to different viral yields. We conclude that human strains studied in this paper may be useful tools in the characterization of molecular determinants involved in viral cytopathogenicity.

Adult↗
PubMed2000

Proteomics characterization of abundant Golgi membrane proteins.

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Amino Acid Sequence↗
PubMed2000

PRINTS-S: the database formerly known as PRINTS.

The PRINTS database houses a collection of protein family fingerprints. These are groups of motifs that together are diagnostically more potent than single motifs by virtue of the biological context afforded by matching motif neighbours. Around 1200 fingerprints have now been created and stored in the database. The September 1999 release (version 24.0) encodes approximately 7200 motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. In addition to its continued steady growth, we report here several major changes to the resource, including the design of an automated strategy for database maintenance, and implementation of an object-relational schema for more efficient data management. The database is accessible for BLAST, fingerprint and text searches at http://www.bioinf.man.ac. uk/dbbrowser/PRINTS/

Database Management Systems↗
PubMed2000

Training Hungarian primary health care teachers: the relevance of a UK postgraduate course for health educators.

OBJECTIVES: This study constituted a formative evaluation of the relevance of the MSc course to the needs of Hungarian primary health care educators. DESIGN: A qualitative, naturalistic approach using in-depth interviews was used to construct the meaning of the experience of the MSc for the Hungarian participants. Interviews were triangulated using observation and documentary analysis. SETTING: The University of Exeter's Institute of General Practice. SUBJECTS: Eight Hungarian primary health care professionals. RESULTS: The evaluation data revealed that the attitude of the Hungarian students to their role as medical educators had been substantially changed by exposure to western models of adult education. There were a number of 'clashes of expectation' between the Hungarian students and the course staff in relation to the course requirements. Reconciliation of these differing expectations required a sequence of ongoing adjustments to the course content and delivery. CONCLUSIONS: Existing postgraduate courses for health educators can accommodate the needs of medical teachers from countries who are developing their primary health care education systems. Successful accommodation is facilitated by ensuring an adequate preparation in relation to language fluency, academic requirements of the course, familiarization with modern approaches to adult education as well as with the local health care delivery system.

Education, Medical, Undergraduate↗
PubMed2000

Zanamivir susceptibility monitoring and characterization of influenza virus clinical isolates obtained during phase II clinical efficacy studies.

Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log(10) 50% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates (n = 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

Animals↗
PubMed1999

In vitro selection and characterisation of influenza B/Beijing/1/87 isolates with altered susceptibility to zanamivir.

We describe the in vitro selection and characterisation of virus derived from B/Beijing/1/87 passaged in the presence of zanamivir. During zanamivir passage, the phenotype of virus isolates was either drug dependent or drug resistant in plaque reduction assays. The susceptibility of the neuraminidase of the drug-dependent isolates was unchanged from that of the wild-type enzyme. The drug-dependent isolates contained two mutations in the viral haemagglutinin: V90A, close to the proposed secondary sialic acid-binding site, and L240Q, close to the primary sialic acid-binding site. Virus isolates that were drug resistant contained the same mutations in the haemagglutinin but also contained the mutation E116G in the neuraminidase. For the drug-dependent viruses, zanamivir susceptibility could not be measured because plaque numbers increased with increasing drug concentration. The in vitro zanamivir susceptibility of drug-resistant viruses was lower than that of the wild-type virus by a factor of 275- to >2532-fold. Neuraminidase containing the E116G mutation has a 33-fold lower affinity for zanamivir than the wild-type enzyme. The finding that the same haemagglutinin mutations are found in both drug-dependent and drug-resistant viruses confirms that the same changes to the receptor binding function can contribute to both phenotypes. This observation demonstrates the interplay between the influenza virus haemagglutinin and neuraminidase in escape from zanamivir inhibition in vitro.

Animals↗
PubMed1999

PRINTS prepares for the new millennium.

PRINTS is a diagnostic collection of protein fingerprints. Fingerprints exploit groups of motifs to build characteristic family signatures, offering improved diagnostic reliability over single-motif approaches by virtue of the mutual context provided by motif neighbours. Around 1000 fingerprints have now been created and stored in PRINTS. The September 1998 release (version 20.0), encodes approximately 5700 motifs, covering a range of globular and membrane proteins, modular polypeptides and so on. The database is accessible via the DbBrowser Web Server at http://www.biochem.ucl.ac.uk/bsm/dbbrowser /. In addition to supporting its continued growth, recent enhancements to the resource include a BLAST server, and more efficient fingerprint search software, with improved statistics for estimating the reliability of retrieved matches. Current efforts are focused on the design of more automated methods for database maintenance; implementation of an object-relational schema for efficient data management; and integration with PROSITE, profiles, Pfam and ProDom, as part of the international InterPro project, which aims to unify protein pattern databases and offer improved tools for genome analysis.

Amino Acid Sequence↗
PubMed1999

A novel clan of zinc metallopeptidases with possible intramembrane cleavage properties.

Computer-based database searching and protein multiple sequence alignment has identified a novel clan of zinc metallopeptidases, which, by phylogenetic analysis, has been shown to contain six subfamilies. The family is characterized by four common transmembrane segments and three conserved sequence motifs. The combination of topology analysis and motif identification has detected three potential Zn2+ coordinating residues. Only two of the sequences of this novel zinc metallopeptidase clan possess any functional annotation, one of which is able to cleave its substrate within a cytosol/transmembrane segment junction. A number of observations suggest that the remaining members of this novel clan may also cleave their substrates within transmembrane segments.

Animals↗
PubMed1998

JavaShade: multiple sequence alignment box-and-shading on the World Wide Web.

RESULTS: JavaShade is a multiple sequence alignment box-and-shade tool for generating high quality printed output that uses a variety of methods for boxing and shading, allowing the most appropriate functions to be chosen for displaying the most meaningful positions in an alignment. AVAILABILITY: JavaShade is available from the WWW at http://industry.ebi.ac.uk/JavaShade

Algorithms↗
PubMed1998

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit.

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.

Antibodies, Monoclonal↗
PubMed1995

Engineered anti-CD38 monoclonal antibodies for immunotherapy of multiple myeloma.

Multiple myeloma is a malignancy of plasma cells for which there is no effective treatment. To develop an immunotherapeutic agent, we have raised a high affinity mAb (AT13/5) against CD38, one of the few well-characterized surface Ags present on myeloma cells. Since murine monoclonals have many disadvantages as human therapeutics, we prepared two engineered forms of the Ab: a CDR-grafted humanized IgG1 and a chimeric FabFc2 (mouse Fab cross-linked to two human gamma 1 Fc). To retain affinity in the humanized Ab, a number of changes were required to the human framework regions of the heavy chain. In particular, through systematic mutagenesis and computer modeling, we identified a critical interaction between the side chains of residues 29 and 78, which may be important for the humanization of other Abs. The properties of the humanized IgG1 and FabFc2 constructs were compared in a series of in vitro tests. Both constructs efficiently directed Ab-dependent cellular cytotoxicity against CD38-positive cell lines, but C was activated only poorly. Neither construct caused down-modulation of CD38, nor did they affect the NADase activity of CD38. Despite their differing structures, both Abs showed similar activity in most assays, although the humanized IgG1 was more potent at inducing monocyte cytotoxicity. These data represent the first direct comparison of CDR-grafted and chimeric FabFc2 forms of the same Ab, and offer no support for the perceived advantages of the FabFc2. These Abs show promise for therapy of multiple myeloma and other diseases involving CD38-positive cells.

ADP-ribosyl Cyclase↗
PubMed1995

Detection of rare allelic variants of the interferon-alpha 2 gene in human genomic DNA.

We have analyzed human donor DNA for the presence of sequences corresponding to allelic variants of the IFN-alpha 2 locus. Using both restriction enzyme digestion of PCR-amplified fragments and sequence analysis of these fragments, we have identified the three reported allelic variants, IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c, in genomic DNA derived from donors of African or Afro-Caribbean origin. This is the first report of the IFN-alpha 2a and IFN-alpha 2c alleles occurring in human donor DNA and supports the view that these are variants of the predominant IFN-alpha 2b allele rather than arising from mutations occurring in cultured cells.

Alleles↗
PubMed1994

Interferon (IFN)-alpha 2 genotype analysis of Chinese chronic hepatitis B patients undergoing recombinant IFN-alpha 2a therapy.

Sixteen Chinese chronic hepatitis B virus (HBV)-infected patients were treated with recombinant interferon-alpha 2a (rIFN-alpha 2a). Of these, 8 made a response to IFN, with titers of neutralizing antibody of 141-4525 as determined by an antiviral neutralization bioassay. To determine whether the immunogenicity of the IFN was directly linked to the patients' genotype, their genomic DNA was analyzed for the presence of the human IFN-alpha 2a gene. None of the patients possessed the gene for IFN-alpha 2a, but only 50% developed neutralizing antibodies. The hypothesis, therefore, of a direct link between antibody formation and genotype cannot be sustained. Alternative explanations of the immunogenicity of IFN-alpha 2a must be sought.

Adult↗
PubMed1993

Rescue, expression, and analysis of a neutralizing human anti-hepatitis A virus monoclonal antibody.

A human anti-hepatitis A virus mAb was rescued from a hybridoma cell line by conventional cDNA cloning, and expressed in CHO cells. The full nucleotide sequences of the mAb H and L chains were determined, revealing a VHI/V lambda II V region combination. Comparisons with germline V genes suggest that the V regions had undergone somatic mutations characteristic of an Ag-driven immune response. A comparison of the binding to hepatitis A virus between mAb derived from the CHO cells and the original hybridoma cell line using ELISA, radioimmunoprecipitation, and solid-phase competition RIA, indicated that the CHO cell-derived mAb fully retained the specificity of the mAb produced by hybridoma cells. Analysis of viral neutralization using a radioimmunofocus inhibition assay demonstrated the retention of antibody functionality after expression in CHO cells, demonstrating the use of this technique in the rescue and high level expression of unstable efficacious human mAb.

Amino Acid Sequence↗
PubMed1993

A humanized CD18 antibody can block function without cell destruction.

Leukocyte integrins are intimately involved in transient adherence of leukocytes to endothelium and to each other in the processes of extravasation and cell activation. In this study, seven mAb directed against human CD11a and two mAb directed against human CD18, the alpha- and beta-chains of the leukocyte functional Ag-1 molecule, respectively, were analyzed for their ability to inhibit several leukocyte functional Ag-1-mediated interactions. The best blocking mAb in these studies, a rat anti-human CD18, YFC51.1, was subsequently humanized by complementarily-determining region grafting, associated with human C regions and expressed. The humanized mAb was shown to maintain binding for human CD18. Even though the humanized mAb was an IgG1 isotype it still retained the functional blocking characteristics of the rat mAb while failing to mediate cell killing. The IgG1 mAb was unable to bind human Clq and could block but did not mediate antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1992

Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

Antibodies, Monoclonal↗