Specificity of human anti-variable heavy (VH ) chain autoantibodies and impact on the design and clinical testing of a VH domain antibody antagonist of tumour necrosis factor-α receptor 1.

During clinical trials of a tumour necrosis factor (TNF)-R1 domain antibody (dAb™) antagonist (GSK1995057), infusion reactions consistent with cytokine release were observed in healthy subjects with high levels of a novel, pre-existing human anti-VH (HAVH) autoantibody. In the presence of HAVH autoantibodies, GSK1995057 induced cytokine release in vitro due to binding of HAVH autoantibodies to a framework region of the dAb. The epitope on GSK1995057 was characterized and dAbs with reduced binding to HAVH autoantibodies were generated; pharmacological comparability was determined in human in-vitro systems and in-vivo animal experiments. A Phase I clinical trial was conducted to investigate the safety and tolerability of the modified dAb (GSK2862277). A significant reduction in HAVH binding was achieved by adding a single alanine residue at the C-terminus to create GSK2862277. Screening a pool of healthy donors demonstrated a reduced frequency of pre-existing autoantibodies from 51% to 7%; in all other respects, GSK2862277 and the parent dAb were comparable. In the Phase I trial, GSK2862277 was well tolerated by both the inhaled and intravenous routes. One subject experienced a mild infusion reaction with cytokine release following intravenous dosing. Subsequently, this subject was found to have high levels of a novel pre-existing antibody specific to the extended C-terminus of GSK2862277. Despite the reduced binding of GSK2862277 to pre-existing HAVH autoantibodies, adverse effects associated with the presence of a novel pre-existing antibody response specific to the modified dAb framework were identified and highlight the challenge of developing biological antagonists to this class of receptor.

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.

Immunoglobulin complementarity-determining region grafting by recombinant polymerase chain reaction to generate humanised monoclonal antibodies.

We describe an approach to rapidly generate humanised monoclonal antibodies by grafting rodent complementarity-determining regions onto human immunoglobulin frameworks using recombinant polymerase chain reaction (PCR) methodology. The approach was applied to grafting a rat complementarily-determining region onto a human framework and amplifying the entire humanised heavy chain. The terminal oligodeoxyribonucleotide primers incorporated restriction sites to allow forced cloning into plasmid vectors for sequencing and expression. No nucleotide errors were introduced into the 1463-bp sequence even after sequential applications of PCR.

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.

Cloning and analysis of the gene encoding the 230-kilodalton merozoite surface antigen of Plasmodium yoelii.

The complete nucleotide sequence of the gene for the 230-kDa precursor to the major merozoite surface antigens (PMMSA) of Plasmodium yoelii YM has been determined. A single open reading frame of 5316 bp encodes a protein of calculated molecular mass 197,000. The deduced amino acid sequence contains potential signal peptide and membrane anchor sequences of 19 and 18 residues, respectively, and a region of six tandemly repeated tetrapeptides, Gly-Ala-Val-Pro. Comparison with the 195-kDa Plasmodium falciparum analogue (Pf195) at the amino acid level indicated an overall homology of approximately 30%, with areas of as high as 60% conservation. The tripeptide repeats present near the N-terminus of the Pf195 antigen are absent from the Py230 sequence. The PMMSA can be divided into 22 blocks based upon interspecies amino acid conservation.

Cloning and sequence analysis of kappa and gamma cynomolgus monkey immunoglobulin cDNAs.

One gamma heavy chain and 10 kappa light chain cynomolgus monkey (Macaca fascicularis) immunoglobulin cDNAs have been cloned and sequenced. Comparisons of the variable (V) regions to human antibody sequences have revealed extensive identity, exhibiting 93% at the amino acid level for the VH framework regions, and 88-99% for the V kappa frameworks. Identification of very few cynomolgus monkey-specific framework region residues suggests a role for cynomolgus monkey antibodies as donators of variable regions to chimeric monoclonal antibodies for utilisation in human therapy with human constant (C) regions. The cynomolgus monkey C kappa region exhibited 83% amino acid identity to its human counterpart, and the C gamma region was 95, 93, 95, and 95% similar to the human C gamma 1, C gamma 2, C gamma 3, and C gamma 4 regions, respectively. Evolutionary analysis of the C gamma genes, using the silent molecular clock, suggests that the divergence between cynomolgus monkey and human occurred before the time at which the ancestral gamma gene diverged into the multiple isotypes observed in humans.

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.

A joint course for general practitioner and practice nurse trainers.

An experimental multidisciplinary course for prospective general practitioner and practice nurse trainers is described. Factual knowledge and attitudes were measured before and after the course and some of the changes measured emphasized the importance of multidisciplinary training. The ideas generated by the group of nurse trainers in terms of their future professional development were identified.

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

Somatic reversion of P transposable element insertion mutations in the singed locus of Drosophila melanogaster requiring specific P insertions and a trans- acting factor.

Destabilization in somatic cells of P-element insertions in the X-linked singed gene of Drosophila melanogaster has been studied. We have shown that some but not all unstable P-element insertions in singed can form mosaics. The cause of this variation is not clear from studies of the restriction maps of the mutations tested. The transposable element movements occur early in development and require, in addition to an appropriate P-element insertion in singed, a trans-acting maternal effect component. Movements appear to occur preferentially in attached-X stocks. However, the maternal effect component maps to the central region of chromosome 2.

General practitioners and their learning styles.

Continuing medical education sessions are often poorly attended by general practitioners. One reason may be that these traditionally consist of lectures by hospital consultants with a strong theoretical bias which may have little relevance to the learning needs of general practitioners. To compare the learning styles of teachers and learners in general practice, learning style questionnaires were administered to 50 hospital clinical tutors, 78 general practitioner trainers, 63 trainees and 47 non-trainer principals. The questionnaire covered four different learning preferences: activist, reflector, theorist and pragmatist. The findings showed that the learning styles of hospital tutors and general practitioner trainers were statistically significantly different to those of non-trainer principals and trainees. The tutors and trainers scored much higher on theorist styles and to a lesser extent on reflector and pragmatist styles. There were no significant differences on activist scores. Since teachers tend to teach in their preferred learning style, which may not match the style of the recipients, these findings have implications for continuing medical education in general practice. These implications are discussed.

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.

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.

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.

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.

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.

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.

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.

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/