Search PubMed⌕ Search

Biomedical subjects

R W Tindle

Publications and source records attributed to R W Tindle.

At least 37 records · Page 2Linked to original sources
PubMed1995

T-helper epitopes of the E7 transforming protein of cervical cancer associated human papillomavirus type 18 (HPV18).

The presence of T-helper epitopes within the E7 transforming protein of human papillomavirus type 18 (HPV18) was sought using a series of overlapping synthetic 15-20 mer peptides spanning the entire 105 amino acid sequence of this protein. Two H-2k restricted T-helper epitopes were defined, comprising 44VNHQHLPARRA55 and 81DDLRAFQQLF90 as the minimal T proliferative epitopes. Peptides containing these epitopes were able to provide cognate help to B epitopes from HPV18E7 protein for production of antibody to this protein in vivo in CBA/CaH mice. No H-2b or H-2d restricted epitopes were demonstrable, and in H-2d mice this was associated with poor antibody response to the E7 protein. There is no "promiscuous" T-helper epitope in HPV18 E7 comparable to the 49DRAHYNI55 sequence in HPV16 E7, and restricted T-helper epitope availability may be a determinant of poor immune responses to this protein after natural infection.

Amino Acid Sequence↗
PubMed1995

Peptide polymerisation facilitates incorporation into ISCOMs and increases antigen-specific IgG2a production.

Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.

Adjuvants, Immunologic↗
PubMed1995

A vaccine conjugate of 'ISCAR' immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants.

TraT protein, known as ISCAR (= Immunostimulatory Carrier), is one of a family of integral membrane proteins (Imps) of Escherichia coli representing powerful carrier molecules which when injected into experimental animals generate substantial antibody and T proliferative responses to molecules conjugated to it. We extend these findings to show that ISCAR functions to stimulate Th1- and Th2-type responses, including specific cytotoxic T cells and tumour protection. We report here that by conjugating to ISCAR a 19mer peptide containing linear B epitopes, a T helper (Th) epitope, and a H-2b-restricted T cytotoxic (CTL) epitope of E7 protein of human papillomavirus type 16 (HPV16), and immunizing C57B1/6 (H-2b) mice, we elicited (i) specific IgG2a and IgG1 antibodies; (ii) IL-2 and IL-4 production by specifically recalled lymph node cells in vitro; (iii) cytotoxic T lymphocytes which specifically killed both E7 peptide-pulsed, and whole E7 gene-transfected tumour target cells; and (iv) in vivo protection against an E7 gene-transfected tumour cell inoculum. These findings have implications for the design of vaccines to stimulate immune responses to endogenously processed target antigens (e.g. tumour-associated antigens) without the unwanted side effects of oil-based adjuvants. In addition they support the case for a E7-targeted therapeutic vaccine for HPV-associated human cervical cancer.

Adjuvants, Immunologic↗
PubMed1994

Chimeric hepatitis B core antigen particles containing B- and Th-epitopes of human papillomavirus type 16 E7 protein induce specific antibody and T-helper responses in immunised mice.

The use of hepatitis B core antigen (HBcAg) as an immunogenic delivery vehicle for foreign epitopes has been reported. Here we report the insertion of DNA sequences encoding immunodominant linear B-epitopes and a "universal" T-helper epitope of human papillomavirus (HPV) type 16 E7 transforming protein into the full-length HBcAg gene cloned into an inducible bacterial expression plasmid (pPN1.0). The resulting chimeric proteins assembled into particles which were highly immunogenic. Mice immunised with particles containing one or two of the three E7 B-epitopes under consideration produced strong epitope-specific antibody responses with both IgG and IgG2a components which recognised eukaryotic E7. T-proliferative responses were elicited to the E7 T-epitope as well as HBcAg T-epitope(s). Lymph node cells from immunised mice produced IL-2 and IL-4 when specifically recalled in vitro, indicating stimulation of both Th1 and Th2 helper cell compartments. Since HBcAg particles can be administered in adjuvant acceptable for human application and can elicit mucosal responses after nasal or oral immunisation, these results have implications for vaccine design in HPV 16-associated anogenital cancer.

Amino Acid Sequence↗
PubMed1994

Human papillomavirus (HPV) type 18 E7 protein is a short-lived steroid-inducible phosphoprotein in HPV-transformed cell lines.

We used a capture ELISA to quantify the E7 protein of human papillomavirus type 18 (HPV-18). In HeLa cells, which express low levels of immunoreactive E7 protein (iE7), iE7 had a mean half-life of 13.5 min. In HPV-18 E7 recombinant baculovirus (E7rec BV)-infected Sf21 cells, which express higher levels of E7, the half-life of iE7 was much longer (90 min and > 24 h, with two different E7rec BVs). For two transformed human cervical cell lines expressing HPV-18 E7, exposure of the cells to hydrocortisone resulted in a twofold increase in steady-state levels of the E7 protein: no similar effect was observed with progesterone, oestrogen or testosterone. The half-life of iE7 was unaltered by hydrocortisone or progesterone exposure. An immunoassay which distinguished Ser33-phosphorylated E7 from E7 not phosphorylated at this residue (Ser33dephospho-E7), showed that in HeLa and Sf21 cells the majority of E7 was phosphorylated: the half-life of both species of E7 was similar in HeLa cells, but the half-life of Ser33dephospho-E7 was much shorter (90 min) in Sf21 cells than that of Ser33phospho-E7 (> 24 h). A HeLa-fibroblast fusion cell line with tumorigenic potential (CGL-1) had a similar ratio of dephospho-E7 to total E7 (0.06), as a similar fusion cell line (CGL-4) with no tumorigenic potential (0.03). We conclude that E7 is a labile phosphoprotein, and that the expression and steady-state level of the E7 protein in eukaryotic cells may be influenced by the hormonal environment of the cells.

Amino Acid Sequence↗
PubMed1993

Expression of human papillomavirus type 16 E7 protein by recombinant baculovirus and use for the detection of E7 antibodies in sera from cervical carcinoma patients.

Although the presence of serum antibodies against the human papillomavirus type 16 (HPV-16) E7 protein has been linked with cervical cancer, currently available assays detect antibodies in only ca. 40% of carcinoma patients. The dependence of these serological assays on synthetic target antigens which present only linear epitopes may be a limiting factor. In order to produce a more realistic target antigen for use in serological assays, we have expressed the HPV-16 E7 protein in insect cells using a recombinant baculovirus vector. Two major E7 forms of ca. 18kDa and 16kDa were produced and characterised. The 16kDa component was shown to be truncated at the N-terminus. A radioimmunoprecipitation assay was developed for the detection of anti-E7 antibodies in human sera. This assay showed a marked increase in detection rate compared with a western blotting method based on bacterially derived E7 fusion proteins.

Amino Acid Sequence↗
PubMed1992

An ELISA capture assay for the E7 transforming proteins of HPV16 and HPV18.

ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.

Animals↗
PubMed1992

Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells.

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.

Antigens, CD↗
PubMed1991

A "public" T-helper epitope of the E7 transforming protein of human papillomavirus 16 provides cognate help for several E7 B-cell epitopes from cervical cancer-associated human papillomavirus genotypes.

We have identified a major T-cell epitope, amino acids 48-54 (DRAHYNI, in one-letter code) in the E7 open reading frame protein of human papillomavirus (HPV) type 16. Lymph node cells from mice immunized with synthetic peptides containing DRAHYNI proliferated and produced interleukin when challenged in vitro with peptide or whole HPV-16 E7 fusion protein. The T epitope was recognized in association with all five major histocompatibility complex class II I-A and I-E alleles tested. Synthetic peptides consisting of DRAHYNI linked to major B-cell epitopes on the E7 molecule formed immunogens capable of eliciting strong antibody responses to HPV-16 E7. The T epitope could provide help for the production of antibody to several B epitopes simultaneously, including a B epitope of HPV-18 E7 protein. Mice immunized with a peptide containing DRAHYNI and B epitope and, at a later date, infected with recombinant vaccinia E7 virus, displayed secondary antibody responses to E7. Because E7 has a role in cell transformation and is the most abundant viral protein in HPV-associated neoplastic cervical epithelial cells, the data have implications for vaccine strategies.

Amino Acid Sequence↗
PubMed1990

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.

Amino Acid Sequence↗
PubMed1990

Identification of B epitopes in human papillomavirus type 16 E7 open reading frame protein.

Human papillomavirus (HPV) type 16 is implicated in the aetiology of anogenital dysplasia which may progress to malignancy. HPV-16 DNA is actively transcribed in cervical carcinomas, the most abundant transcripts being from the E6 and E7 early open reading frames. The E7 protein has been shown to have transforming activity in vitro. In this report we define four immunodominant B epitopes within the protein corresponding to the E7 gene, using a panel of murine monoclonal antibodies. Three epitopes are linear and lie within the N-terminal region of the molecule, and are unique to the HPV-16 E7 protein. One epitope is non-linear and presumed to be conformational. At least three of the four epitopes of the E7 protein are detectable by immunoprecipitation from an HPV-16-infected cervical carcinoma cell line. The demonstrated immunogenicity of the E7 protein allows us to deduce that this molecule may be a potential candidate for incorporation in a vaccine against cervical cancer.

Amino Acid Sequence↗
PubMed1990

Immunology of anogenital human papillomavirus (HPV) infection.

A subgroup of human papillomaviruses (HPVs) is implicated in the aetiology of neoplastic change in anogenital epithelium. Although abundant circumstantial evidence exists for the involvement of the immune system in the control of HPV infection, restriction of infection to epithelium and the lack of a viral productive phase pose problems for immune response induction, and for immunological effector mechanisms. In this article we discuss how HPV antigens may be presented to the immune system on the surface of keratinocytes as well as, or in addition to, presentation by 'classical' antigen presenting cells, and outline putative roles for non-specific and specific (B- and T-cell) effector functions. We discuss the prospects for a vaccine and the use of anti-HPV antibodies in immunodiagnosis.

Antigen-Presenting Cells↗
PubMed1987

Expression of HPCA-1 and HLA-DR antigens on growth factor- and stroma-dependent colony forming cells.

The expression of HLA-DR and HPCA-1 antigens (recognized by the L243 and BI.3C5 antibodies respectively) on adult human bone marrow cells was examined by fluorescence activated cell sorting and colony assays. Nearly all the (day 14) lineage restricted and multipotential colony forming cells analysed in methylcellulose cultures in the presence of added growth factors express HLA-DR and HPCA-1 determinants. Two colour cell sorting reveals that the lineage restricted HLA-DR positive progenitors express variable levels of BI.3C5 positivity whereas most of the multipotential progenitors, the multi-CFC or CFU-GEMM, are highly BI.3C5 positive. The isolated HLA-DR and BI.3C5 positive populations also contain haemopoietic precursors which adhere to and form colonies on pre-formed stromal layers. Thus, haemopoietic progenitors assayed in both types of culture system can be analysed and enriched by simultaneous two-colour sorting using anti-HLA-DR and BI.3C5 monoclonal antibodies. Similarities in the antigenic phenotype of such cells, however, precludes the use of these reagents for segregating growth factor-dependent from stroma-dependent progenitors.

Adult↗
PubMed1986

Platelet-derived growth factor stimulates growth of highly enriched multipotent haemopoietic progenitors.

Platelet-derived growth factor (PDGF) has been shown to stimulate growth of normal and malignant fibroblasts, glial cells and smooth muscle cells. A growth promoting effect on human haemopoietic precursors has also been described, but the interpretation of this haemopoietic proliferative response to PDGF has been hampered by the lack of purity of the target population. In this study we show that PDGF promotes growth of early bone marrow haemopoietic progenitors depleted of either monocytes or T lymphocytes which are known to influence haemopoiesis. Moreover, the action of PDGF is even increased on a highly enriched BI-3C5 early bone marrow population. BI-3C5 is a novel monoclonal antibody which recognizes an antigen present on all multilineage colony-forming cells (CFU-mix) (Tindle et al. 1985). BI-3C5 positively and negatively sorted fractions were obtained by fluorescence activated cell sorting (FACS) and PDGF was found to stimulate growth of CFU-mix in the BI-3C5-positive fraction (consisting of only 4-6% of the marrow population), the effect being more marked than that on unsorted bone marrow. The results suggest that the product of the cellular proto-oncogene c-sis (the putative structural gene for the beta chain of PDGF) may play a regulatory role in the in vivo proliferation of multipotent haemopoietic progenitors.

Adult↗
PubMed1985

A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow.

A MCA raised against the human acute myelogenous leukaemia cell line KG1 reacted with only KG1 among 26 haematopoietic cell lines covering the major lineages. It reacted with early myeloid (M1/2), 1 of 2 acute myelomonocytic (M4) and most non-B non-T leukaemias, including blast crises of CGL. Among M1-AML cells, both MPO+ and MPO- blasts were BI-3C5+. Blasts in 3 Tdt+ M1-AMLs were simultaneously BI-3C5+. BI-3C5 reacted with 4% cells in normal BM, many of which were histologically recognisable as myeloid precursors. 8-15% of BI-3C5+ cells in BM were simultaneously Tdt+, and all were weakly Ia antigen+. BI-3C5 was unreactive with all peripheral leucocytes, with M3 and M5 AMLs, with lymphoid and myeloid leukaemias of "mature" phenotype (T-ALL, B-ALL, CLL, CGL) and with non-haematopoietic cell lines. BI-3C5 precipitated a 120K moiety from 125I-labelled KG1 membranes. It was not blocked by J5 anti-cALLA. The potential use of BI-3C5 in the classification of acute leukaemias is discussed.

Animals↗
PubMed1985

Variation in myc gene amplification and expression in sublines of HL60 cells.

Three sublines of the human promyelocytic leukaemia cell line HL60 have been isolated. Histochemical, karyotypic and immunological analyses have confirmed the identity of the three sublines as HL60. However, the extent to which c-myc homologous DNA sequences are amplified in the genomes of these cells varies from 4- to 30-fold. Moreover, the relative abundance of c-myc-related RNA sequences in uninduced cells of each subline is directly proportional to the gene copy number. It is concluded that, while high levels of the c-myc gene and its transcripts may have had a role in the establishment of HL60 cells, these are not required for maintenance of the HL60 phenotype. However we cannot rule out the possibility that a low level of amplification and enhancement of transcription has a role in maintenance of this phenotype.

Antibodies, Monoclonal↗
PubMed1985

A monoclonal-antibody-based immunoradiometric assay for apoprotein B in low-density lipoprotein.

Five different mouse monoclonal antibodies were raised against apoprotein B, the major protein in human low-density lipoproteins (LDL). Competitive binding studies suggested that these antibodies recognized four different epitopes on the lipoprotein particle. All antibodies bound to very-low-density and intermediate-density lipoproteins, which are the metabolic precursors of LDL and also contain apoprotein B. One antibody (P1C1) displayed a relative specificity for apoprotein B in LDL as compared with that in very-low-density lipoprotein and was used as a radiolabeled first antibody in an immunoradiometric assay for this lipoprotein. Measurements, either on isolated LDL or directly on plasma samples, demonstrated that this assay provides a rapid and precise method of quantification.

Animals↗