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Biomedical subjects

J Hackett

Publications and source records attributed to J Hackett.

At least 37 records · Page 2Linked to original sources
PubMed1999

Monoclonal antibodies to an HIV-1 group O envelope recombinant.

Monoclonal antibodies were developed to a recombinant HIV-I group O envelope protein derived from the isolate HAM112. These monoclonal antibodies were characterized for reactivity to a series of overlapping synthetic peptides (29-30 mers) covering gp120 C-terminal and gp41 ectodomain regions of the HIV-1 group O envelope protein. Most of these monoclonal antibodies reacted with peptides spanning sequences analogous to HIV-1 group M epitopes identified from studies in mice and humans. However, several of the antibodies that were nonreactive to individual peptides did react to a mixture of longer peptides from the N-terminal and C-terminal helical regions of the gp41 ectodomain. The monoclonal antibodies described in this study are valuable tools for characterization of antigenic differences between HIV-1 group O and group M viruses.

Amino Acid Sequence↗
PubMed1999

Accelerated filament formation from tau protein with specific FTDP-17 missense mutations.

Tau is the major component of the neurofibrillar tangles that are a pathological hallmark of Alzheimers' disease. The identification of missense and splicing mutations in tau associated with the inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 demonstrated that tau dysfunction can cause neurodegeneration. However, the mechanism by which tau dysfunction leads to neurodegeneration remains uncertain. Here, we present evidence that frontotemporal dementia and Parkinsonism linked to chromosome 17 missense mutations, P301L, V337M and R406W, cause an accelerated aggregation of tau into filaments. These results suggest one mechanism by which these mutations can cause neurodegeneration and frontotemporal dementia and Parkinsonism linked to chromosome 17.

Alzheimer Disease↗
PubMed1998

A hypermutable insert in an immunoglobulin transgene contains hotspots of somatic mutation and sequences predicting highly stable structures in the RNA transcript.

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

Animals↗
PubMed1998

Somatic hypermutation of an artificial test substrate within an Ig kappa transgene.

We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the Vkappa167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, Vkappa167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer's patch-derived or immunized splenic B220+ cells with high peanut agglutinin levels. Two derivative lines of Vkappa167/EPS were made, Vkappa167/POX and Vkappa167/PEPS. While none of the Vkappa167/POX transgenic lines demonstrated mutation, the Vkappa167/PEPS transgene was highly mutated in B220+ splenic B cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable Vkappa167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.

Animals↗
PubMed1998

Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.

Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), historically termed Pick's disease. Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics. Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q21.11; the tau gene also lies within this region. We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon 10. The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10. This causes more frequent usage of the 5' splice site and an increased proportion of tau transcripts that include exon 10. The increase in exon 10+ messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17.

Alternative Splicing↗
PubMed1998

Detection and differentiation of HIV-1 group O sera from HIV-1 group M and HIV-2 using recombinant antigens and peptides.

Recombinant antigens and peptides were used to develop an HIV slot immunoblot assay to confirm and differentiate infection by HIV-1 group M, HIV-1 group O or HIV-2. Recombinant antigens from the gag, pol or env regions of HIV-1 and HIV-2, in addition to synthetic peptides from the immunodominant region (IDR) of transmembrane proteins gp41 (HIV-1) or gp36 (HIV-2), were blotted on nitrocellulose strips and used as a substitute for competitive Western blots. Evaluation of a large number of samples (N = 440) from various regions of the world, using the immunoblot, showed effective differentiation of HIV-1 group M, HIV-1 group O and HIV-2. The immunoblot identified correctly all (24/24) HIV-1 group O samples that were confirmed subsequently by PCR and sequence analysis. The immunoblot is a useful tool for identifying HIV-1 group O seropositive samples and has the potential to identify other serological HIV variants that may represent detection problems for HIV screening assays using HIV-1 group M subtype B reagents.

Amino Acid Sequence↗
PubMed1998

Cis-acting sequences that affect somatic hypermutation of Ig genes.

We review our studies on the mechanism of somatic hypermutation of immunoglobulin genes. Most experiments were carried out using Ig transgenes. We showed in these experiments that all required cis-acting elements are present within the 10-16 kb of a transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region. Several Ig gene enhancers are permissive for somatic mutation. Association of the enhancer with its natural Ig promoter is not necessary. However, the mutation process seems specific for Ig genes. No mutations were found in housekeeping genes from cells with high levels of somatic hypermutation of their Ig genes. The Ig enhancers may provide the Ig gene specificity. An exception may be the BCL6 gene, which was mutated in human but not in mouse B cells. Transcription of a region is required for its mutability. When the transcriptional promoter located upstream of the variable region is duplicated upstream of the constant region, this region also becomes mutable. This suggests a model in which a mutator factor associates with the RNA polymerase at the promoter, travels with the polymerase during elongation, and causes mutations during polymerase pausing. The DNA repair systems, nucleotide excision repair and DNA mismatch repair, are not required. Our recent data with an artificial substrate of somatic mutation suggest that pausing may be due to secondary structure of the DNA or nascent RNA, and the specific mutations to preferences of the mutator factor.

Animals↗
PubMed1998

Recombinant mouse-human chimeric antibodies as calibrators in immunoassays that measure antibodies to Toxoplasma gondii.

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Amino Acid Sequence↗
PubMed1998

Immunoglobulin transgenes as targets for somatic hypermutation.

This review describes studies on somatic hypermutation of immunoglobulin genes that were started in the mid-80s in collaboration with Ralph Brinster. Almost all of the experiments were carried out using Ig transgenes as targets for the somatic mutation mechanism. Ig transgenes can be very good targets of somatic mutation, despite many different transgene integration sites. Thus, the required cis-acting elements must be present within the approximately 10 kb of the transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region in unmanipulated sequences. Several Ig gene enhancers are permissive for somatic mutation and they do not have to be associated with the Ig promoter they normally interact with. However, the mutation process does seem to be specific for Ig genes. No mutations were found in several housekeeping genes isolated from cells that had very high levels of somatic hypermutation of their Ig genes. This suggests that the Ig enhancers provide the lg gene specificity. An exception is the Bcl-6 gene, encoding a transcription factor, which was found to be mutated in normal human memory B cells. When the transcriptional promoter that is located upstream of the variable region is duplicated upstream of the constant region, this region is mutated as well. This suggests a transcription coupled model in which a mutator factor associates with the RNA polymerase at the initiation of transcription, travels with the polymerase during elongation, and causes mutations during polymerase pausing. Our recent data with an artificial substrate for somatic mutation suggest that the mutations are increased by increased stability of the secondary structures in the nascent RNA, and the specific nucleotides that are mutated are due to preferences of a mutator factor.

Animals↗
PubMed1997

Molecular cloning, nucleotide sequence, and function of a site-specific recombinase encoded in the major 'pathogenicity island' of Salmonella typhi.

The genome of the typhoid fever bacterium, Salmonella typhi, contains at least three large insertions ('pathogenicity islands') relative to the chromosome of Salmonella typhimurium (which is normally non-invasive for humans) [Liu, S.-L., Sanderson, K.E., 1995. Rearrangements in the genome of the bacterium Salmonella typhi. Proc. Natl. Acad. Sci. USA 92, 1018-1022]. DNA encoding a site-specific recombinase (the rci gene) and an adjacent putative pilus-tip adhesin protein (the pilV gene) was located (near min 94) in the major 'pathogenicity island' of the S. typhi chromosome, cloned, and sequenced. It was shown that the Rci protein inverted a DNA segment of 490 bp, between two 19-bp inverted repeat elements, to place either of two possible C-termini on a constant N-terminal region of the PilV protein. Both possible PilV proteins were seen when the alternative pilV genes were transcribed from the T7 promoter and translated in vivo. Both the rci and the N-terminal region of the pilV gene show a high degree of homology to genes encoded by the IncI2 plasmid R721 and the IncI1 plasmid R64. One of the possible pilV C-termini (in the pilV1 gene) is highly homologous to shufflon C (one of the possible PilV C-termini) of R64; the other possible pilV C-terminus (in the pilV2 gene) shows no homology to any published shufflon. In the R64 plasmid, the PilV proteins are pilus-tip adhesins; different PilV proteins recognize different potential recipient bacterial strains as a prelude to mating in liquid culture [Komano, T., Kim, S.-R., Yoshida, T., Nisioka, T., 1994. DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64. J. Mol. Biol. 243, 6-9]. It is likely that S. typhi encodes pili bearing two alternative PilV proteins as tip adhesins, one of which recognizes, specifically, a membrane component of Escherichia coli K-12, while the specificity of the other PilV protein is not known.

Amino Acid Sequence↗
PubMed1996

Search for retrovirus in patients with multiple sclerosis.

Multiple sclerosis (MS) patient samples were screened for known or novel retroviruses using an ultrasensitive technique, IMx PERT, that detects the presence of reverse transcriptase (RT). This procedure has 10(5)- to 10(7)-fold greater sensitivity than conventional RT assays and is capable of detecting 10 to 50 virions. Moreover, IMx PERT is at least as sensitive as polymerase chain reaction, and requires no previous knowledge of viral nucleotide sequence. The MS specimens analyzed in this study included 136 sera from 79 patients and 128 cerebrospinal fluid samples from 53 patients with relapsing or chronic progressive disease. In addition, peripheral blood mononuclear cells from 19 MS patients were cultured in an attempt to amplify or induce expression of low-copy number or cell-associated retrovirus. No evidence of retrovirus was found in any of the specimens obtained from MS patients.

Base Sequence↗
PubMed1996

The mechanism of somatic hypermutation studied with transgenic and transfected target genes.

Somatic hypermutation of Ig genes is beginning to be understood in molecular terms. Ig transgenes have served as model test genes and been shown to mutate, just as endogenous genes, with a peak of mutation over the VJ region and a sparing of the C region. The levels of somatic mutation appear to be related to the expression of the transgenes. DNA hypermethylation of modified Ig transgenes interferes with both expression and somatic hypermutation. Test substrates consisting of bacterial lacZ alpha or supFtRNA inserted within x transgenes were shown to be rescuable as expressible bacterial plasmids, but did not seem to be targeted. A synthetic sequence consisting of alternating restriction enzyme sites, that cannot be subject to methylation, was found to be a reliable transgenic substrate for easy assay of somatic mutation. The generation of a transgenic mouse with a x transgene in which the transcriptional promoter has been duplicated upstream of the C region has given new clues to the mutation mechanism. In this transgene, transcripts initiate from both the C region and the V region promoters, and both regions, but not the sequences between them, are hypermutated. These results suggest that somatic hypermutation is linked to the initiation of transcription. A model is proposed in which somatic mutation is dependent on transcription coupled DNA repair.

Animals↗
PubMed1996

Paediatric rheumatology: clinical practice review. Physiotherapy and occupational therapy for juvenile chronic arthritis: custom and practice in five centres in the UK, USA and Canada

Physiotherapy and occupational therapy are widely accepted as being of central importance for the treatment of juvenile chronic arthritis (JCA). However, these approaches have rarely been subject to critical scrutiny. The aims of this report are to highlight some of the inter-centre similarities and differences observed in the implementation of physical and occupational therapy for JCA, and to emphasize the need for scientifically controlled research in this area. During a series of visits to several paediatric rheumatology units in the UK, USA and Canada, three aspects of the service were noted: treatment philosophy, physical interventions used for the treatment of JCA and quality-of life and independence training activities. There was general consensus with the philosophy that early physical intervention was a vital part of the treatment plan for JCA, although all therapists were concerned that compliance with treatment modalities was poor. Differences between units in the approach to acute arthritis, the use of foot othoses and wrist splints, the treatment of joint contractures and the use of general quality-of-life training activities were noted. Although it was widely recognized that controlled research into the efficacy of physical intervention was needed, no centre had a co-ordinated plan for such investigations. Keywords: Juvenile chronic arthritis, Physiotherapy, Occupational therapy

Journal Article↗
PubMed1996

Physiotherapy and occupational therapy for juvenile chronic arthritis: custom and practice in five centres in the UK, USA and Canada.

Physiotherapy and occupational therapy are widely accepted as being of central importance for the treatment of juvenile chronic arthritis (JCA). However, these approaches have rarely been subject to critical scrutiny. The aims of this report are to highlight some of the inter-centre similarities and differences observed in the implementation of physical and occupational therapy for JCA, and to emphasize the need for scientifically controlled research in this area. During a series of visits to several paediatric rheumatology units in the UK, USA and Canada, three aspects of the service were noted: treatment philosophy, physical interventions used for the treatment of JCA and quality-of-life and independence training activities. There was general consensus with the philosophy that early physical intervention was a vital part of the treatment plan for JCA, although all therapists were concerned that compliance with treatment modalities was poor. Differences between units in the approach to acute arthritis, the use of foot orthoses and wrist splints, the treatment of joint contractures and the use of general quality-of-life training activities were noted. Although it was widely recognized that controlled research into the efficacy of physical intervention was needed, no centre had a co-ordinated plan for such investigations.

Arthritis, Juvenile↗
PubMed1994

Locus-specific analysis of human leukocyte antigen class I expression in melanoma cell lines.

Surface expression of human leukocyte antigen (HLA) class I antigens on melanoma lines was evaluated by locus-specific monoclonal antibodies (mAbs) with three different techniques: Fluorescence-activated cell sorting (FACS), immunohistochemistry with cytospin preparation (ICP), and complement-mediated cytotoxicity (CMC). Eleven HLA class I-expressing cell lines developed from metastases were used. Specific expression of HLA loci was examined under routine culture conditions and after 48-h incubation in interferon-gamma (IFN-gamma; 500 U/ml). Loss of allelic expression was seen in one line (586-MEL): Products of genes coding for HLA-A29 and -B44, in strong linkage disequilibrium, were not detectable. HLA-A antigens were consistently detected by all methodologies and minimally affected by pretreatment with IFN-gamma. HLA-B antigens were detectable in 8 of 11 lines by ICP and 3 of 11 lines by CMC. By FACS the supratypic specificity HLA-Bw6 was expressed at low levels in most lines (mean fluorescence 47.2 +/- 13.4 and rose to 259.8 +/- 45.9 after incubation with IFN-gamma; p < 0.001). HLA-Cw antigen detection by CMC correlated with HLA-B (p < 0.01), suggesting that down-regulation and sensitivity to IFN-gamma are shared by the two loci. This low expression of the HLA-B antigens may play a role in the evasion of the host immune response and its up-regulation may be useful in allowing tumor antigen recognition.

Cytotoxicity Tests, Immunologic↗