Induction of Bcl-xL-specific cytotoxic T lymphocytes in mice.

The induction of active immunity against tumour-associated antigens to prevent relapse of cancer is a promising approach but has so far shown only low efficacy. This low efficacy may in part be due to clonal escape of tumour cell variants by the downregulation of antigen expression or inflammation-induced dedifferentiation. Identification of novel tumour-associated antigens that at the same time are essential for continued tumour cell survival is thus critical for the development of active cancer vaccinations. At the same time, identification of novel endogenous murine tumour antigens will help improve preclinical development of cancer immunotherapy. The anti-apoptotic protein Bcl-xL has been suggested to be such an essential tumour antigen, but the lack of well-defined murine epitopes have delayed preclinical studies of Bcl-xL-targeting cancer vaccines. Here, we report the identification of two novel murine tumour-associated epitopes TAYQSFEQV and AFFSFGGAL derived from mouse Bcl-xL. Dendritic cell (DC)-based vaccination induced CD8(+) T cells capable of producing IFN-γ upon restimulation with these epitopes. Thus, our data may benefit the design of future immunotherapy strategies by providing a preclinical model for cancer vaccination with an endogenous tumour antigen that can be combined with other cancer treatments.

Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer.

BACKGROUND: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. METHODS: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case-control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. RESULTS: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. CONCLUSION: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection.

Early detection of cancer in the general population: a blinded case-control study of p53 autoantibodies in colorectal cancer.

BACKGROUND: Recent reports from cancer screening trials in high-risk populations suggest that autoantibodies can be detected before clinical diagnosis. However, there is minimal data on the role of autoantibody signatures in cancer screening in the general population. METHODS: Informative p53 peptides were identified in sera from patients with colorectal cancer using an autoantibody microarray with 15-mer overlapping peptides covering the complete p53 sequence. The selected peptides were evaluated in a blinded case-control study using stored serum from the multimodal arm of the United Kingdom Collaborative Trial of Ovarian Cancer Screening where women gave annual blood samples. Cases were postmenopausal women who developed colorectal cancer following recruitment, with 2 or more serum samples preceding diagnosis. Controls were age-matched women with no history of cancer. RESULTS: The 50 640 women randomised to the multimodal group were followed up for a median of 6.8 (inter-quartile range 5.9-8.4) years. Colorectal cancer notification was received in 101 women with serial samples of whom 97 (297 samples) had given consent for secondary studies. They were matched 1 : 1 with 97 controls (296 serial samples). The four most informative peptides identified 25.8% of colorectal cancer patients with a specificity of 95%. The median lead time was 1.4 (range 0.12-3.8) years before clinical diagnosis. CONCLUSION: Our findings suggest that in the general population, autoantibody signatures are detectable during preclinical disease and may be of value in cancer screening. In colorectal cancer screening in particular, where the current need is to improve compliance, it suggests that p53 autoantibodies may contribute towards risk stratification.

Partial vapor-phase hydrolysis of peptide bonds: A method for mass spectrometric determination of O-glycosylated sites in glycopeptides.

In this study we present a method for determination of O-glycosylation sites in glycopeptides, based on partial vapor-phase acid hydrolysis in combination with mass spectrometric analysis. Pentafluoropropionic acid and hydrochloric acid were used for the hydrolysis of glycosylated peptides. The reaction conditions were optimized for efficient polypeptide backbone cleavages with minimal cleavage of glycosidic bonds. The glycosylated residues were identified by mass spectrometric analysis of the hydrolytic cleavage products. Although glycosidic bonds are partially cleaved under acid hydrolysis, the resulting mass spectra allowed unambiguous determination of the glycosylation sites. Examples are shown with mannosyl- and mucin-type glycopeptides. Performing the hydrolysis in vapor eliminates the risk for contamination of the sample with impurities from the reagents, thus allowing analysis of the reaction products without further purification both by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus.

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.

Substrate specificities of three members of the human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, -T2, and -T3.

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.

Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B.

Human saliva contains high and low molecular weight mucin glycoproteins, that are distinct. Recently the gene encoding low molecular weight salivary mucin was cloned and designated MUC7, whereas the primary structure of high molecular weight salivary mucin is unclear. Furthermore, the expression patterns of high and low molecular weight salivary mucins in salivary glands have been debated. We have previously generated monoclonal antibodies specific for the peptide cores of salivary mucins. In the present study a monoclonal antibody specific for high molecular weight salivary mucin was used to screen a human salivary gland cDNA library. A single clone, SAL1, was identified and found to be encoded by tracheobronchial mucin gene MUC5B. A previously reported partial cDNA sequence from salivary mucin was linked to SAL1/MUC5B by genomic cloning and reverse transcriptase-polymerase chain reaction. Northern analysis of salivary gland RNA probed with SAL1 suggested that MUC5B was highly expressed in salivary glands. In situ hybridization was performed with a SAL1/MUC5B probe and a MUC7 probe. All mucous cells from the submandibular, sublingual, palatine, and labial glands labeled with the MUC5B probe, while serous cells labeled with the MUC7 probe. These findings were in accordance with our previous immunohistological results of the cellular localizations of salivary mucins. The results suggest that MUC5B is identical to or a major fraction of high molecular weight salivary mucin, and that MUC5B is expressed in all mucous cells of salivary glands. In contrast MUC7 is expressed in serous cells of salivary glands except the parotid glands.

Loss of a novel mucin-like epithelial glycoprotein in oral and cervical squamous cell carcinomas.

Stratified squamous epithelia of oral and cervical mucosa express high levels of simple mucin-type O-linked carbohydrates, and these are known to undergo structural changes in relation to epithelial differentiation and neoplastic transformation. O-glycans in these epithelia are associated with the cell membrane, but the identity of the carrier molecule(s) remains largely unknown. We report here the identification of a membrane-bound M(r) 200,000-250,000 glycoprotein (gp230) that is expressed in stratified squamous epithelia of the oral cavity. Western blot analysis identified gp230 as a major carrier of simple-mucin type carbohydrate antigens in buccal nonkeratinized mucosal epithelium, suggesting that it may represent a mucin-like molecule. A monoclonal antibody PANH4 defining a protein epitope of gp230 was generated. The PANH4 epitope was localized by immunohistology to suprabasal cell layers of buccal epithelium and was also found in larynx, esophagus, vagina, and exocervix, but not in epidermis. Data showed that gp230 was distinct from MUC1 or CD44. It is interesting that in most cases gp230 was not expressed in squamous cell carcinomas of buccal and cervical mucosa. A few moderately differentiated carcinomas, mainly from cervix, expressed the gp230 epitope. The results suggest that a membrane-bound mucin-like molecule, gp230, is associated with the differentiated phenotype of normal mucosal stratified squamous epithelia and that expression of gp230 generally is lost in severe oral epithelial dysplasia and squamous cell carcinomas of oral and cervical mucosa.

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal nature of fibroblasts. Cells were first cultured in DMEM with 0.5% fetal calf serum (FCS) and then incubated for 8 h, and 72 h in fresh DMEM with 10% FCS. Total RNA was isolated and used for Northern blotting with a P32-labeled KGF cDNA probe. Total RNA from cultured keratinocytes was used as negative controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% FCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore, RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in noncultured periodontal ligament cells.

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. Identification and separation of two distinct transferase activities.

Using a defined acceptor substrate peptide as an affinity chromatography ligand we have developed a purification scheme for a unique human polypeptide, UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc-transferase) (White, T., Bennett, E.P., Takio, K., Sørensen, T., Bonding, N., and Clausen, H. (1995) J. Biol. Chem. 270, 24156-24165). Here we report detailed studies of the acceptor substrate specificity of GalNAc-transferase purified by this scheme as well as the Gal-NAc-transferase activity, which, upon repeated affinity chromatography, evaded purification by this affinity ligand. Using a panel of acceptor peptides, a qualitative difference in specificity between these separated transferase activities in four rat organs and two human organs also revealed qualitative differences in specificity. The results support the existence of multiple Gal-NAc-transferase activities and suggest that these are differentially expressed in different organs. As the number of GalNAc-transferases existing is unknown, as is the specificity of the until now cloned and expressed GalNAc-transferases (T1 and T2), it is as yet impossible to relate the results obtained to specific enzyme proteins. The identification of acceptor peptides that can be used to discriminate GalNAc-transferase activities is an important step toward understanding the molecular basis of GalNAc O-linked glycosylation in cells and organs and in pathological conditions.

Effect of truncal vagotomy and pylorotomy on gastric emptying.

The effect of transthoracic vagotomy and a conventional pylorotomy on gastric emptying of a hyperosmolar glucose meal was evaluated in four dogs by means of an isotope technique. The measurements were made preoperatively and 1 month and 3-6 months after each operation. Vagotomy significantly increased the initial rate of gastric emptying, whereas the total fraction of the meal emptied in 60 min was the same as before operation. The pylorotomy did not alter the emptying. Approximately 50% of the meal was still retained in the stomach at the end of the tests. The abnormal gastric emptying of the glucose meal after vagotomy was normalized by drinking an 'aperitif' of 20% soya bean oil 15 min before the ingestion of the glucose meal. It is concluded that a vagotomy alters the pattern but not the 60-min fraction of gastric emptying of a liquid meal, whereas a pylorotomy alters neither the pattern nor the 60-min fraction of gastric emptying. The vagally denervated gastrointestinal tract has maintained mechanisms for control of the gastric emptying.

Pathophysiology of massive trauma with therapeutic implications.

In addition to its local effects, severe trauma has several consequences for the whole organism. Partly, these are hormonal, humoral and neural regulatory mechanisms - negative feedback mechanisms to regain homeostasis, a balance of the "milieu intérieur". Blood loss and disturbances in the fluid compartments is one of the central pathological features. The shock period is accompanied by mobilization of defense mechanisms and energy. The length and extent of of the catabolic phase depends on the extent of the injury, the effectiveness of the therapeutic efforts and the disturbances of organ and system functions. Repair proceeds gradually aiming at anatomical and functional restitution.

Measurement of duodeno-gastric reflux in dogs.

For the elaboration of a formula estimating the reflux volume of duodenal secretion into the stomach, gastric and duodenal secretion have been studied during pentagastrin and secretin stimulation. In the stimulated gastric secretion the concentration of sodium varied with the secretion rate, whereas the concentration of chloride was nearly constant. The concentration of sodium in duodenal juice was constant, but the chloride concentration dropped significantly during secretin stimulation. Secretin induces duodenal reflux. When duodenal reflux occurs, a substantial amount of the sodium in gastric juice is attributable to intruding duodenal juice. The formula gives the duodenal reflux volume in 15-min samples on the basis of the sodium in gastric secretion and in the duodenal juice. A concentration of sodium in samples of gastric juice twice exceeding the concentration of sodium in maximally stimulated gastric secretion (8 +/- 2 mmol/l, mean +/- S.D.) may suggest 'contamination' of the samples with duodenal juice. The present formula allows for quantitative determination of this 'contamination.'

The puzzle of blood platelets in extracorporeal circulation. Comments based on recirculation experiments.

Oozing of blood from wound tissues after some time of total body perfusion and the demonstration of microemboli have brought into focus the inevitable disappearance of platelets from the blood during extracorporeal circulation. Recirculation experiments using dog blood are reported. The effect of a bubble oxygenator and a membrane oxygenator is compared. It is concluded that in principle they both contribute to the removal of functionally active platelets.