Expression and purification of prostate-specific membrane antigen in the baculovirus expression system and recognition by prostate-specific membrane antigen-specific T cells.

Antigen-specific immunotherapy of cancer depends on a consistent source of well-defined protein antigen. Production of recombinant protein offers the obvious solution to this problem but few comparisons of recombinant and native proteins in cellular immune assays have been reported. We report expression of a putative immunotherapy antigen, prostate-specific membrane antigen (PSMA), in insect cells using a baculovirus vector. T cells stimulated with recombinant PSMA or native PSMA derived from the LNCaP cell line recognized both native PSMA and recombinant, baculoviral PSMA. These data indicate that PSMA produced in Sf9 cells is immunologically cross-reactive with native PSMA and therefore suitable for immunotherapy as it is recognized by both cellular and humoral immune responses.

Report of immune monitoring of prostate cancer patients undergoing T-cell therapy using dendritic cells pulsed with HLA-A2-specific peptides from prostate-specific membrane antigen (PSMA).

BACKGROUND: In this paper we describe our program for the immune monitoring of phase II participants given dendritic cell (DC)/prostate-specific membrane antigen (PSMA)-based immunotherapy, and we also present some initial findings. METHODS: Phase II subjects received six administrations of autologous dendritic cells exogenously pulsed with two peptides derived from PSMA. Prior to the initial infusion, and following each treatment, peripheral blood mononuclear cells (PBMC) were collected for the generation of dendritic cells as well as for comprehensive immune monitoring. RESULTS: Thus far, an increase in PSMA-peptide-specific as well as overall cellular reactivity has been observed in several patients receiving DC plus PSM-P1 and -P2, as measured by delayed-type hypersensitivity (DTH) test and enzyme-linked immunosorbant assay (ELISA). CONCLUSIONS: Our initial observations using an ELISA and DTH test indicate that we are enhancing cellular immunity in prostate cancer patients following infusion with DC plus PSMA-derived peptides. Several methods are underway to comprehensively monitor both cell-mediated and humoral immune responsiveness, including: determining anti-PSMA serum antibody titers, testing immunogen-restricted responder-cell proliferation and cytotoxicity, assessing aberrations in signal transduction, antigen processing, and presentation, and measuring soluble factors that may promote tumor outgrowth.

Cancer immunotherapy for prostate cancer.

Our approach to prostate cancer immunotherapy involves two components dendritic cells as antigen-presenting cells; and the antigen used to target T-cell attack, HLA-A0201-associated peptides from prostate specific membrane antigen (PSMA). We have conducted a phase I dose-ranging study in 51 men with advanced prostate cancer, using dendritic cells pulsed with a PSMA peptide. no significant toxicity was observed. In that study, T-cell response was enhanced, with seven men meeting NCPC and PSA criteria for partial response. We are now conducting a phase II study with 67 men, who will receive 6 infusions of dendritic cells that have been pulsed with 2 PSMA peptides, at 6-week intervals. The phase II study design and rationale is described in this paper.

Uptake of drugs from topically applied anti-inflammatory preparations applied to racing animals.

OBJECTIVE: To determine whether a drug detected in the blood or urine of a racing animal could have penetrated through the skin from a topically applied preparation. DESIGN: Blood and urine of dogs and horses were analysed after topical administration of three common nonsteroidal anti-inflammatory preparations. EXPERIMENTAL METHOD: Dimethylsulphoxide was analysed using a gas chromatograph with a flame photometric detector. Phenylbutazone, its metabolites and lignocaine were analysed using a gas chromatograph with a mass selective detector. RESULTS: Dimethylsulphoxide, phenylbutazone and lignocaine were detected in dog urine after multiple applications of the preparations. The maximum concentration of dimethylsulphoxide in dog urine correlated with the concentration of dimethylsulphoxide in the preparation. Phenylbutazone penetrated the skin more effectively from the cream than from the solution or gel preparations. This penetration was independent of the concentration of dimethylsulphoxide. CONCLUSION: The superior penetration of phenylbutazone from the cream can be explained by it being present as a neutral molecule in an hydrophobic medium. It is proposed that phenylbutazone penetrates the skin of greyhounds most effectively by a hydrophobic lipid route which is likely to be different from the path by which dimethylsulphoxide penetrates the skin.

Glucose tolerance and insulin sensitivity in ponies and Standardbred horses.

The existence of an innate insulin insensitivity in ponies was investigated and compared with the situation in larger breeds of horse. Ponies that were fat or had previously suffered laminitis were found to be far more intolerant to oral glucose loading (1 g/kg bodyweight [bwt]) than normal ponies or Standardbreds. These ponies also exhibited a far greater response in plasma insulin levels after glucose loading. Insulin response tests (0.4 iu/kg bwt insulin intravenously) showed only a minimal and very protracted response in both the fat and laminitic groups. The relevance of these findings in regulation of carbohydrate and lipid metabolism, and their role in the pathogenesis of hyperlipaemia, are discussed.

Essential fatty acids in the fetal and newborn lamb.

The concentrations of linoleic and linolenic acids and their metabolites in the liver, kidney, brain, erythrocytes and plasma of fetal lambs at various stages of gestation, and of newborn and 2-week-old suckled lambs was determined. Throughout gestation the fetal tissues, erythrocytes and plasma all contained low levels of linoleic and linolenic acids together with consistently high levels of their long-chain polyunsaturated metabolites. The triene: tetraene (eicosa-5,8,11-trienoic acid/arachidonic acid) ratio was always 0.4 or less except at birth when it reached 0.6 in liver and 0.9 in plasma. Milk intake significantly increased the linoleic and linolenic acid levels in the lamb by 2 weeks after birth. These results show that the developing fetal lamb should not be regarded as being deficient in essential fatty acids, as suggested by previous investigators. It is proposed that the total metabolites of linoleic and linolenic acids are the most appropriate measure of the essential fatty acid status of the fetal lamb.

Essential fatty acid deficiency and evidence for arachidonate synthesis in the cat.

1. There is controversy regarding the capacity of the cat to convert 18: 2 omega 6 to 20: 4 omega 6 and the ability of the essential fatty acid (EFA)-deficient cat to produce 20: 3 omega 9. 2. This paper reports the isolation and identification of 20: 3 omega 9 from kidney phospholipids of EFA-deficient cats. 3. The results suggest that the cat is capable of limited synthesis of 20: 4 omega 6 using a delta 5- and delta 8-desaturase.

Metabolism of linoleic acid in the cat.

Cats fed a diet containing linoleate as the only polyunsaturated fatty acid showed extremely low levels of arachidonate in the plasma lipids, as well as an increase in linoleate, eicosadienoate and an unknown fatty acid. Administration of [1-14C]linoleic acid and [2-14C]eicosa-8,11,14-trienoic acid to cats showed that in the liver there was no conversion of the [1-14C] 18:2 to arachidonate, whereas there was significant metabolism of [2-14C] 20:3 to arachidonate. It was found when methyl-gamma-linolenate was fed to cats that the level of 20:3 omega 6 and 20:4 omega 6 in the erythrocytes increased significantly. These results show that there is no significant delta 6 desaturase activity in the cat, whereas chain elongation and delta 5 desaturase enzymes are operative. The unknown fatty acid was isolated from the liver lipids and shown to be a 20-carbon fatty acid with 3 double bonds and which by gas liquid chromatography could be separated from 20:3 omega 9 and 20:3 omega 6. The presence of the delta 5-desaturase activity and the results of the ozonolysis studies indicated that this unknown fatty acid was eicosa-5,11,14-trienoic acid.

Oxygen affinity responses to 2,3-diphosphoglycerate, and methaemoglobin formation in horse and human haemoglobins.

The oxygen affinities of horse and human haemoglobins were compared in the absence and presence of the allosteric effector 2,3-diphosphoglycerate (2,3-DPG). Horse haemoglobin solutions showed significantly smaller responses to the presence of 2,3-DPG, and this difference may be due to different amino acid substitutions at position NA2(2)beta. Horse haemoglobin solutions from erythrocytes containing different ratios of the two different haemoglobin types showed similar oxygen affinities in the absence and presence of 2,3-DPG. Horse haemoglobins in solution were found to autoxidise to methaemoglobin much more readily than human haemoglobin under the same conditions, and this is an important consideration when measuring the oxygen affinity of horse haemoglobin solutions. This difference could be due to different amino acid residues at position NA2(2)beta.