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J Ackland

Publications and source records attributed to J Ackland.

12 recordsLinked to original sources
PubMed2005

Recombinant NY-ESO-1 cancer antigen: production and purification under cGMP conditions.

The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.

Antigens, Neoplasm↗
PubMed1999

Induction of specific CD8+ T-lymphocyte responses using a human papillomavirus-16 E6/E7 fusion protein and autologous dendritic cells.

When intracellular viral proteins are degraded, only a limited number of peptide epitopes are capable of eliciting specific CD8+ cellular immune responses for a given human leukocyte antigen (HLA) haplotype. We sought to induce CD8+ T-lymphocyte (CTL) responses to human papillomavirus-16 (HPV-16) E6 and E7 proteins using a recombinant E6/E7 fusion protein and autologous human dendritic cells (DCs). CTLs were generated by in vitro stimulation using a recombinant HPV-16 E6/E7 fusion protein and autologous DCs from a healthy HLA-A*0201 donor. CTL specificity was assessed by cytokine release assays when the cells were reacted with autologous DC targets coincubated with the E6/E7 fusion protein. These CTLs were also reacted with the immunodominant E7 peptides (E711-20 and E7(86-93)) and DCs as a target. As a negative control, DCs were incubated with or without an irrelevant control protein (Helicobacter pylori) as target for the E6/E7-induced CTLs. The E6/E7-induced CTLs were capable of specific recognition of target DCs coincubated with E6/E7 but not the control protein. When E6/E7-specific CTLs were reacted with DCs and either E7(11-20) or E7(86-93), specific peptide recognition was also detected. These data demonstrate that specific CTLs can be elicited using autologous human DCs and a HPV-16 E6/E7 fusion protein. Therefore, extracellular viral proteins seem to be engulfed and processed by DCs; then the immunodominant HLA-A2-restricted peptides become available for CD8+ T-lymphocyte recognition. These data suggest that vaccine strategies using recombinant viral proteins may overcome the limitation of peptide epitopes for specific HLA haplotypes and may, therefore, permit more generalized clinical application.

Antigen Presentation↗
PubMed1991

Expression of ovarian inhibin during pregnancy in the rat.

We have examined the expression of the rat inhibin genes in the maternal ovary during pregnancy. RNA blot analysis indicates that the inhibin-alpha chain mRNA is expressed in the ovary throughout gestation at levels comparable to those observed in cycling rats. In situ hybridization shows that the inhibin-alpha and -beta A mRNAs are produced in the granulosa cells of developing antral follicles; little or no hybridization to functional corpora lutea is observed. Early in pregnancy, a large number of follicles hybridize to both alpha- and beta A-inhibin cDNA probes. Many of these follicles undergo atresia during the first half of pregnancy, and the number of inhibin-expressing follicles reaches a nadir on day 15. This is followed by an increase in inhibin-producing follicles, which peaks just before parturition. The increase in inhibin-expressing follicles observed in late pregnancy corresponds to a small rise in serum inhibin levels, as measured using an alpha chain-specific RIA. After the first postpartum ovulation, few hybridizing follicles are observed. Ovariectomy in either early (day 6) or mid (day 15) pregnancy results in a significant fall in serum inhibin levels and a robust increase in serum FSH levels 9 h after surgery. These results suggest that inhibin is produced by the maternal ovary during pregnancy, that its synthesis is modulated during late gestation, and that inhibin may play a role in regulating FSH secretion during pregnancy.

Animals↗
PubMed1989

Synthesis of small proteoglycans substituted with keratan sulfate by rabbit articular chondrocytes.

35S-Labeled proteoglycans produced by chondrocytes from immature and mature rabbits were fractionated on associative CsCl gradients. In all cultures, greater than 85% of the incorporated radioactivity was present in the A1 fraction (rho 1.60) as chondroitin sulfate/keratin sulfate-substituted aggregating proteoglycan monomer; the remainder was present in small proteoglycans in the A2, A3, and A4 fractions of low buoyant densities (rho 1.53, 1.45, 1.37, respectively). Detailed glycosaminoglycan analysis of the A2, A3, and A4 fractions showed dermatan sulfate-rich species were present throughout. However, in both immature and mature cultures, 30-45% of the glycosaminoglycans in the A3/A4 combined fractions were present as keratan sulfate, as shown by insensitivity to digestion with chondroitinase ABC, specific digestion with endo-beta-galactosidase, and reactivity with antibody 5D4. Immature and mature chondrocytes synthesized very similar amounts of the low buoyant density keratan sulfate proteoglycan on a per cell basis. Moreover, 51 and 37% of the total keratan sulfate produced by immature and mature chondrocytes, respectively, were present in the low buoyant density proteoglycan. Pulse-chase experiments indicated that the low buoyant density keratan sulfate was not derived from the large aggregating proteoglycan by proteolysis in the extracellular space. The small keratan sulfate proteoglycans appear to be present as a species distinct from the small dermatan sulfate proteoglycans in these cultures in that they can be separated on Q-Sepharose chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent size (40-60 kDa), composition, and heterogeneity of the keratan sulfate proteoglycans suggest that they may be related to the small keratan sulfate proteoglycans of cornea.

Animals↗
PubMed1986

Immunochemical and enzymatic analyses of extracts of the house dust mite Dermatophagoides pteronyssinus.

The allergenicity and enzyme content of extracts of Dermatophagoides pteronyssinus whole mite, cuticle (CUT), and spent-growth medium (SGM) used in its culture have been examined. Both direct RAST binding and RAST-inhibition studies demonstrated that the whole mite extract was approximately threefold more potent than either the CUT or SGM extracts. Crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis demonstrated that whole mite extracts contained 36 antigens of which nine were judged to be allergens (Dpt 1, 3, 4, 7, 8, 12, 17, 22, and 31). In contrast, extracts of CUT and SGM both contained 15 mite antigens of which five were judged to be allergens (Dpt 4, 8, 12, 17, and 22). Enzyme studies demonstrated that whole mite extracts from various sources contained esterases, phosphatases, aminopeptidases, and glycosidases. Both the CUT and SGM extracts contained a similar spectrum of enzymes, although the glycosidases were present at relatively low levels. Combined crossed immunoelectrophoresis and enzymic studies identified three antigens (Dpt 6, 13, and 24) with esterase activity, one antigen (Dpt 27) with beta-glucuronidase activity, and one antigen (Dpt 14) with acid phosphatase activity. Thus far, none of these enzymes has been demonstrated to be allergenic. Both enzymic and immunochemical studies suggested that not all mite allergens are of an excretory/secretory origin, but some (e.g., Dpt 1, 3, 4, 7, and 31) are derived from the whole mite per se.

Allergens↗
PubMed1984

Quantitation and thermal stability of the mite allergen DPT 12 in whole mite extracts.

Twelve whole mite extracts (WME) from seven sources were examined for their content of the allergen Dpt 12, protein content, and the inhibitory capacity in RAST by use of paper discs coupled with either WME or Dpt 12. Linear regression analysis demonstrated that the concentration of Dpt 12 determined either by single radial immunodiffusion or by RAST inhibition did not correlate with either the RAST-inhibition titer with use of discs coupled with WME or with protein content, suggesting that Dpt 12 content does not reflect the overall potency of WMEs. However, statistically significant correlations were obtained between Dpt 12 content and whole mite-RAST inhibition titers if extracts from the same manufacturer were examined or if data obtained from extracts containing large Dpt 12 to protein concentrations (greater than 25%) were excluded from the analysis. In contrast, statistically significant correlations between protein concentration and WME-RAST inhibition titers (p = 0.00037) and between Dpt 12 content and Dpt 12 RAST-inhibition titers (p = 0.00001) were found. Thermal stability studies demonstrated that Dpt 12 per se and WMEs were stable at 4 degrees C for up to 3 mo. Storage at 23 degrees C for 3 mo, however, resulted in partial loss of both Dpt 12 and whole-mite activity. Substantial losses occurred at 36 degrees C for both 1-month and 3-month incubation periods. Esterase, esterase lipase, and alkaline phosphatase were found in three WMEs studied. Dpt 12 was biochemically unrelated to these enzymes.

Allergens↗
PubMed1984

Heterogeneous immunocytochemical reactivities of oCRF-41-like material in the human hypothalamus, pituitary and gastrointestinal tract.

Tissue specimens of human hypothalami, pituitaries and gastrointestinal tract were studied with an indirect immunoperoxidase technique using 6 different rabbit ovine corticotrophin-releasing factor (oCRF-41) antisera. All these antisera detected oCRF-41-like immunoreactivity in parvocellular neurones of the paraventricular nucleus of the hypothalamus and their nerve terminals adjacent to portal capillaries in the infundibular stem and posterior pituitary. 1 antiserum recognised oCRF-41-like immunoreactivity in the growth hormone cells of the anterior pituitary. 3 other antisera recognised oCRF-41-like immunoreactivity in mucosal cells of the gastric antrum. Pre-treatment of the antisera with sauvagine did not affect the immunostaining of the hypothalamic and gastric cells, but quenched the immunostaining of growth hormone cells in the anterior pituitary. It is concluded that (1) in human hypothalami a CRF is synthesized that is structurally very similar to oCRF-41; (2) in growth hormone cells of the anterior pituitary oCRF-41-like immunoreactive material can be detected which shares antigenic determinants with sauvagine; (3) in mucosal cells of the gastric antrum oCRF-41-like immunoreactivity is present that is comparable but probably not identical to hypothalamic CRF and is not sauvagine-like.

Amphibian Proteins↗
PubMed1984

Glucose modulation of somatostatin and LHRH release from rat hypothalamic fragments in vitro.

In the rat, hypoglycaemia inhibits growth hormone secretion, but the mechanism is unclear. To investigate this further, we have studied the effects of glucose and 2-deoxy-D-glucose on somatostatin and LHRH release from rat hypothalamic fragments incubated in vitro. Glucose (1.35-22 mM) was added to glucose-free medium and 5 and 50 mM 2-deoxy-D-glucose were added to medium containing 5.5 mM glucose. Medium somatostatin and LHRH levels were measured by RIA. Somatostatin and LHRH released diluted in parallel with synthetic somatostatin and LHRH. Sephadex gel filtration demonstrated two molecular forms of somatostatin, 70% coeluting with somatostatin-14 and 30% with somatostatin-28; LHRH coeluted with synthetic LHRH. KCl (30-100 mM) resulted in release of somatostatin and LHRH; this was reduced in calcium-free medium. Basal and K+-stimulated somatostatin release were significantly increased by reducing glucose levels (r = -0.6, p less than 0.001). Basal LHRH was not influenced by glucose. Basal and K+-induced somatostatin release were significantly increased by 2-deoxy-D-glucose (p less than 0.05), while LHRH levels remained unchanged. Our results demonstrate that basal and K+-induced somatostatin release from rat hypothalamic fragments are modulated by local glucose concentrations, and this effect is specific as it is not paralleled by LHRH changes. We suggest that the reduction in growth hormone secretion during hypoglycaemia in the rat might be mediated, at least in part, via a direct effect of glucose on somatostatin release.

Animals↗
PubMed1983

Characterisation of immunoreactive somatostatin in human fetal hypothalamic tissue.

Levels of immunoreactive somatostatin (IRS) were measured in extracts of hypothalamic tissue from human fetuses of 12-26 weeks gestation. The IRS contents (0.7-22.5 ng) and concentrations (2.7-118.0 pg/mg wet weight tissue) both increased slightly with gestation. Sephadex G-50 chromatography of 11 extracts showed up to four peaks of IRS, one co-eluting with synthetic somatostatin-14 (S14), a second co-eluting with synthetic somatostatin-28 (S28) and two other peaks having approximate molecular weights of 6000 and 10 000, respectively. The levels of S14 and S28 increased significantly during gestation, while the levels of 6000 molecular weight IRS decreased with age. We suggest that the increase in S14 and S28 levels may be the cause of the fall in circulating growth hormone (GH) in the fetus in later gestation.

Female↗
PubMed1982

Stability of pro-opiocortin-related peptides in post-mortem mouse brain tissue.

The stability of pro-opiocortin-related peptides, growth hormone and somatostatin was investigated in mouse brain for up to 72 h post mortem. The peptide content in acid extracts of whole mouse brain was measured by radioimmunoassay and the molecular forms characterised by chromatography on Sephadex G-50 under acid-dissociating conditions. The brain content of the pro-opiocortin-related peptides and growth hormone rose markedly with time after death and chromatography showed this to be due to an increase in the same molecular forms present immediately post mortem, with no significant peptide fragmentation or precursor cleavage. These rises were not seen in the brains of those animals hypophysectomized at death. In contrast, the levels of somatostatin were not significantly altered post mortem though there was a shift in the relative distribution of immunoactivity between the different molecular forms. These results indicate that the peptide content of post-mortem mouse brain may be affected by leakage of pituitary contents and that even though the levels of other neuropeptides may not change, their molecular forms may be altered.

Adrenocorticotropic Hormone↗
PubMed1980

Magnetizable solid-phase fluoroimmunoassay of thyroxine by a sequential addition technique.

We describe a simple fluoroimmunoassay for the determination of thyroxine concentrations in serum. The method, "sequential addition, separation fluoroimmunoassay," involves both thyroxine labeled with fluorescein and magnetizable cellulose/iron oxide particles to which antibodies to thyroxine have been covalently linked. Serum sample or standard is incubated with an excess of the solid-phase antibody; the particles, which now carry most of the antigen in the sample, are sedimented onto a magnet and the supernate, which contains endogenous fluorophores and other interfering factors, is removed and discarded. Excess labeled thyroxine is then added, and, after incubation, the fluorescence in the supernate (free fraction), which is related directly to the amount of thyroxine in the sample is measured. For the whole procedure, including fluorometry, each sample is treated entirely within disposable polystyrene test tubes. Correlation studies with two different radioimmunoassays showed good agreement.

Fluoresceins↗