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R J Booth

Publications and source records attributed to R J Booth.

At least 37 records · Page 2Linked to original sources
PubMed1990

The mapping of an antibody-binding region on the Mycobacterium tuberculosis 19 kilodalton antigen.

To localize the epitopes of four independently derived murine mAb IT-10, IT-12, IT-16, and IT-19 on the 19-kDa Ag protein of Mycobacterium tuberculosis, expression plasmids were constructed containing deletions of the gene encoding the 19-kDa protein. Reaction of the 4 mAb with Western blots of the truncated recombinant proteins revealed two epitope specificities in the recognition of the 19-kDa protein. IT-10 was found to be dependent only on the presence of amino acids surrounding the first cysteine residue, whereas IT-12, IT-16, and IT-19 all required the presence of both the first and third cysteine residues. These two cysteine residues are separated by 135 amino acids, and are considered to be brought together by tertiary folding of the protein to form an assembled epitope for IT-12, IT-16, and IT-19. These three mAb demonstrated differing sensitivities to the modification of reduced 19-kDa protein using iodoacetamide: a treatment that should have prevented the reformation of disulfide bonds within the protein. This result suggests that, although IT-12, IT-16, and IT-19 appear to be specific for the same epitope, there are probably fine-specificity differences in this recognition. IT-10 was not sensitive to the absence of disulfide bonds within the 19-kDa protein, suggesting that the epitope is not conformationally sensitive, and is likely to be linear in nature.

Antigens, Bacterial↗
PubMed1990

The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae.

A DNA probe encoding approximately 80% of the 18-kDa protein gene of Mycobacterium leprae was isolated and tested for specificity by assessing hybridization of the probe to genomic DNA from taxonomically related and unrelated DNA samples. The 360-base-pair (bp) probe was specific for M. leprae DNA and did not hybridize with genomic DNA from 18 species of bacteria nor with DNA from human, murine, and armadillo sources. Oligonucleotide primers were synthesized corresponding to the 5' and 3' ends of the 360-bp fragment to yield a fragment of similar size on amplification of M. leprae DNA by the polymerase chain reaction (PCR). A simple procedure for DNA extraction from M. leprae-infected tissues was developed that provided suitable template DNA for amplification. The PCR test was specific for M. leprae DNA from human and murine sources and detected M. leprae DNA in biopsies from leprosy patients and from control and uninfected human skin biopsy preparations seeded with as few as 100 M. leprae.

Animals↗
PubMed1989

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay.

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).

Amino Acid Sequence↗
PubMed1989

Characterization of an antibody-binding epitope from the 18-kDa protein on Mycobacterium leprae.

A murine mAb, designated L5, appears to be specific for an epitope on a protein from Mycobacterium leprae of restricted distribution within the mycobacteria. This protein, of Mr 18,000 (18 kDa) is of interest because monoclonal antibodies raised against it do not appear to cross-react with other mycobacterial pathogens. The L5 antibody-binding epitope has been mapped by two complementary methods; expression of gene fragments and synthesis of short peptides. This L5-binding region of the 18-kDa protein (amino acids 109 to 115) shows some homology to a region of the GroEL heat shock family of proteins. Characterization of this antibody-binding epitope may lead to a reagent of use in early diagnosis of infection.

Animals↗
PubMed1989

Homology of the 70-kilodalton antigens from Mycobacterium leprae and Mycobacterium bovis with the Mycobacterium tuberculosis 71-kilodalton antigen and with the conserved heat shock protein 70 of eucaryotes.

Two lambda gt11 recombinant clones, JKL2 and JKL15, each containing an insert coding for part of the highly immunogenic 70-kilodalton (kDa) protein antigen, were isolated from a Mycobacterium leprae genomic library by immunoscreening with the monoclonal antibody L7. Clone JKL2 contained the largest insert, 2.3 kilobase pairs. Nonoverlapping fragments of this insert were used as probes and showed strong hybridization to a number of Mycobacterium tuberculosis-lambda gt11 recombinants producing proteins recognized by an anti-M. tuberculosis 71-kDa monoclonal antibody, IT11. One clone from a recombinant Mycobacterium bovis library was also characterized by using L7, and the insert from this clone, B5bt, hybridized strongly to the M. leprae probes as well. The nucleotide sequence of the 1,037-base-pair coding region of the JKL2 M. leprae clone which encodes the carboxy-terminal half of the 70-kDa protein had extensive homology with genes from a number of species. In all cases, these genes, including the recently described Ag63 and Ag361 of Plasmodium falciparum, were found to be members of the heat shock protein 70 (hsp 70) family of genes. At the amino acid level, homology was maximal between amino acids 83 through 107 and 159 through 184, which showed extreme conservation (92 and 85% identity) with Escherichia coli DnaK amino acids 386 through 409 and 460 through 485, respectively, and was 51% homologous over the entire coding region (amino acids 1 through 344 of JKL2). In contrast, amino acids 129 through 158 had maximal homology with the phylogenetically more distant Xenopus laevis hsp70. Homology declined substantially in the carboxy-terminal 34 amino acids. The predicted ATP-binding functional activity of the 70-kDa antigen from M. bovis was confirmed with affinity purification of the antigen by binding to ATP-agarose and elution with ATP. In view of the conservation of sequences between these mycobacterial antigens and mammalian endogenous cellular enzymes, further evaluation of these molecules in vivo may aid in understanding tolerance to self-antigens as well as provide potentially useful immunodiagnostic reagents.

Adenosine Triphosphate↗
PubMed1988

Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein.

Recombinant clones expressing antigenic determinants of the 18-kDa protein antigen from Mycobacterium leprae recognized by the L5 monoclonal antibody were isolated from a lambda gt11 expression library and their nucleotide sequences determined. All clones expressed the M. leprae-specific determinant as part of a large fusion protein with Escherichia coli beta-galactosidase. The deduced amino acid sequence of the coding region indicated that all the lambda gt11 recombinant clones contained an incomplete M. leprae gene sequence representing the carboxy-terminal two-thirds (111 amino acids) of the 18-kDa gene and coding for a peptide of m.w. 12,432. Subsequent isolation and sequencing of a 3.2kb BamHI-PstI DNA fragment from a genomic M. leprae cosmid library permitted the deduction of the complete 148 amino acid sequence with a predicted m.w. of 16,607. A second open reading frame 560 bases downstream from the 18-kDa coding sequence was found to code for a putative protein of 137 amino acids (m.w. = 15,196). Neither this nor the 18-kDa amino acid sequence displayed any significant homologies with any proteins in the GENBANK, EMBL, or NBRF data bases. Crude lysates from recombinant lambda gt11 clones expressing part of the 18-kDa protein have been reported to stimulate the proliferation of some M. leprae-specific helper T cell clones. Thus, it is significant that the complete 18-kDa sequence contains five short peptides predicted to be possible helper T cell antigenic epitopes based on their propensity to form amphipathic helices. Although three of these occur within the 111 amino acid carboxy-terminal peptide expressed by lambda gt11 clones, the most highly amphipathic peptide is found in the amino-terminal region not present in the lambda gt11 recombinants.

Amino Acid Sequence↗
PubMed1988

The use of a 'universal' yeast expression vector to produce an antigenic protein of Mycobacterium leprae.

This report describes the use of a recombinant yeast expression vector to synthesize and secrete the Mycobacterium leprae 18 kDa antigenic protein. The protein is secreted with a short hydrophilic 'flag' octapeptide fused to its amino-terminus. The fusion protein can be purified directly from yeast culture supernatant through an anti-flag antibody affinity column and the flag octapeptide removed using enterokinase. The method provides a simple and rapid means of obtaining recombinant 18 kDa antigen in quantities suitable for immunological studies.

Antigens, Bacterial↗
PubMed1988

Expression of functional human interleukin-2 receptors in murine interleukin-3-dependent cells.

A murine recombinant retrovirus containing the cDNA encoding the human p55 interleukin-2 (IL2)-binding protein was used to insert this gene into a murine interleukin-3 (IL3)-dependent cell line, FD.C/1. Virus-infected cells, maintained in medium supplemented with IL3, expressed human p55 on the cell surface and readily adapted to growth using human IL2. In the presence of human IL2, the synthesis of the endogenous murine p55 binding protein was induced in FD.C/1 cells, making it difficult to determine whether the human p55 protein was actively involved in the process of growth signal transduction. A cloned cell line, FD.huIL2R-2, was identified which grew in the presence of human IL2 but which had lost the ability to synthesize murine p55 protein. Growth of this clone was inhibited by the monoclonal antibody 2A3 which specifically blocked binding of human IL2 to the human p55 binding protein. Analysis of restriction enzyme digests of FD.huIL2R-2 cell DNA revealed that a rearrangement of a murine p55 gene had occurred, implying that virus infection had resulted in the integration of retroviral DNA at a site close to or within a murine p55 gene. If IL2 signal transduction involves binding to a surface heterodimeric receptor for IL2, it is argued that FD.huIL2R-2 cells contain an IL2 receptor complex of murine p70 and human p55 IL2-binding proteins. Alternatively, it is possible that integration of human p55 DNA into a site close to a murine p55 gene may lead to a hybrid p55 IL2-binding protein. If FD.huIL2R-2 cells express murine p70 IL2-binding protein as part of the receptor complex, the inability of cells to grow in murine IL2 implies that IL2 binding to p70 protein alone is insufficient for a growth signal in these cells. FD.huIL2R-2 cells grow at rates similar in IL3- or human IL2-dependent states. It is likely therefore that the biochemical pathways that control each of these lymphokine-dependent growth states are very similar.

Animals↗
PubMed1987

Autologous blood transfusion for major vascular surgery using the Sorenson Receptal Device.

Twenty-five patients having aortic surgery had blood scavenged using the Sorenson Receptal Device (Group A) and were compared with twenty-five patients having homologous blood transfusion (Group H). Mean intraoperative blood loss was similar in both groups, Group A 3224 (SD 2392) ml, Group H 2999 (SD 1579) ml, but the mean homologous blood replacement was significantly different intraoperatively, Group A 1.2 (SD 1.7) units, Group H 2.7 (SD 1.8) units. Total intra-hospital homologous blood replacement was not significantly different, Group A 4.0 (SD 3.4) units, Group H 5.5 (SD 5.8) units. Mean haemoglobin concentration in the scavenged blood was 8.5 (SD 2.1) g/dl compared to 10.8 (SD 2.4) g/dl in the median aged homologous blood units crossmatched for Group H. Mean red cell half life in the scavenged blood was the same as that for the homologous blood, 24 (SD 5) days, but plasma-free haemoglobin and bacterial contamination was greater in the scavenged blood. There was no difference in the incidence of postoperative renal dysfunction, coagulopathy or mortality between the two groups of patients.

Aged↗
PubMed1985

T cell-replacing factor in specific antibody responses to influenza virus by human blood B cells.

In man, B cell maturation factors obtained from T cells or T cell lines have been shown to induce antibody formation in mitogen or anti-immunoglobulin activated B cells, and in some continuous B cell lines, but the relationships between these factors and B cell differentiation factors in antigen-specific antibody responses is unclear. We have now shown that supernatants from phytohemagglutinin-activated tonsil cells, or from the Gibbon Ape T cell line MLA-144, can substitute for T cells in the specific antibody response by human blood B cells to influenza virus. Thus, T cell-depleted non-rosette-forming (E-) cells prepared from peripheral blood mononuclear cells made antibody when cultured with antigen and factor together, whereas control cultures of E- cells with either antigen or factor alone did not. Moreover, E- cells cultured with factor and influenza virus strain A/X31 made antibody to A/X31, but not the non-cross-reacting strain, B/HK (and vice versa) showing that the response was antigen specific. The activity in these supernatants, therefore, fulfilled the functional definition of T cell-replacing factor (TRF). The possibility that interleukin 2 (IL 2) present in the TRF-containing supernatants was expanding residual T cells in the E- preparations to provide normal T cell help was excluded in three different ways. First, E- cells depleted of T (Leu4+) cells to undetectable levels made normal amounts of antibody when cultured with antigen and TRF. Secondly, a limiting dilution technique was employed to show that help in cultures of E- cells and TRF was not mediated by antigen-specific T helper cells. Thirdly, TRF-containing supernatants depleted of IL2 retained activity, whereas purified IL2 was inactive. Preliminary purification of TRF by gel filtration on Ultrogel AcA54 columns showed that all the activity eluted in a single peak between 35 000 and 43 000 molecular weight. This distinguishes human TRF from IL 2 and from other B cell maturation factors with a molecular weight range of 15 000-20 000 which act on continuous B cell lines. In addition to TRF, supernatants from phytohemagglutinin-activated tonsils also contained a factor which could induce polyspecific IgM production, but only in cultures containing significant numbers of T cells. This additional activity may have been due to IL 2, and provides an explanation for the apparent T cell-dependent effects sometimes observed in experiments designed to test B cell differentiation factors on T cell-depleted normal B cells.

Antibodies, Viral↗
PubMed1985

Induction of antibody responses to influenza virus in human lymphocyte cultures. I. Role of interleukin 2.

The in vitro T cell-dependent antibody response of human lymphocytes to influenza virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.

Antibodies, Monoclonal↗
PubMed1984

Interleukin 1 induces proliferation in two distinct B cell subpopulations responsive to two different murine B cell growth factors.

Two distinct classes of B cell growth factor (BCGF) have been described. BCGF I induces proliferation of anti-IgM-activated B cells, whereas BCGF II acts on a subpopulation activated by dextran sulfate. We examined the effect on both these B cell subsets of a B cell growth and differentiation factor that copurifies with interleukin 1 (BGDF/IL 1) produced by the WEHI-3 cell line. Proliferation was induced by BGDF/IL 1 in cultures of normal spleen cells depleted of T cells and adherent cells and activated by anti-IgM or dextran sulfate. Little effect was observed in cultures of spleen cells from B cell-deficient CBA/N mice. In cultures of normal B cells activated with anti-IgM and dextran sulfate together, BGDF/IL 1 induced a proliferative response comparable with the sum of the responses to the factor and either stimulus alone. These results suggest that anti-IgM and dextran sulfate activate distinct and independent B cell subpopulations, both of which are responsive to BGDF/IL 1.

Animals↗
PubMed1984

Interleukin-2 is required for the induction of erythrocyte--specific antibody responses in human lymphocyte cultures.

The involvement of interleukin-2 (IL-2) in the generation of human antibody responses was studied using the pokeweed mitogen (PWM)-induced sheep erythrocyte-specific plaque-forming cell (SE-PFC) response of cultured tonsilar lymphocytes. IL-2-containing supernatants from phytohemagglutinin-stimulated human spleen or tonsil cells or the cloned human T cell line JURKAT were capable of replacing the requirement for PWM in the generation of SE-PFC and resulted in a more prolonged response than that observed with PWM. This effect of IL-2-containing medium was dependent on the presence of SE-derived antigens and was not due to the induction of polyclonal antibody synthesis. When purified by gel filtration the SE-PFC-inducing activity co-eluted with IL-2 with an apparent molecular weight of approximately 15,000 (range, 8,000-20,000). Selective removal of IL-2 from crude or partially purified IL-2 preparations by extensive adsorption with activated murine thymocytes or an IL-2-dependent T cell line completely eliminated SE-PFC-inducing activity, indicating that IL-2 itself was necessary for the effect. The question of whether or not this effect was mediated through T cells is discussed.

Animals↗