Search

Backgound: The major histocompatibility (MHC) class I antigens act as associative molecules for interaction amongst immuno-competent cells. The grooves of class I antigens are normally loaded with self peptides of between 8 and 11 amino acids. However, when the cells transform to malignant state they may carry peptide(s) of non-self origin within these grooves. Using immuno-bead purification followed by high-performance liquid chromatography (HPLC), this study attempted to isolate peptides from class I antigens of various biological specimens Results: The combination of immuno-bead purification (BP) and HPLC was reliable for peptide isolation. Class I antigens and associated peptides could be isolated from normal peripheral blood leukocyte (PBL). Under the same conditions, the PBL yielded almost twice as much peptide as that of tumour cell lines. The HPLC profile of peptides (range of 8–10 amino acid residues) isolated from a bladder and a cervical tumour cell line showed unique features. In the case of the bladder line there were at least 22 peptides. In addition, the class I-associated peptides could also be isolated from kidney tumour fragments of three individuals. In each the isolated peptides showed a unique HPLC peak profile with some similarities as well as differences. Conclusions: These data indicated a variation in the nature of peptides isolated from different specimens. The approach showed the feasibility of preparing peptide(s) from a relatively small number of cells. The data also showed that peptide isolation could also be carried out from tumour tissue biopsies paving the way for the future of peptide vaccination in cancer patients following the identification of putative tumour-specific replicate(s).

Background/Methods: In this study, immunobead purification, dot-blot, immunocytochemical staining, and SDS-PAGE techniques in combination with high-performance liquid chromatography were used to isolate human leukocyte antigen (HLA) class I antigens and associated peptides from a bladder tumour cell line (Fen) before and after gene transfection. Results: The results showed that: (1) Transfection of the class I negative Fen cell line with normal β-microglobulin (β2-m) gene resulted in the restoration of missing class I antigens. (2) The intact class I antigens could be isolated from lysate of the β2-m gene transfected cells using Sepharose CNBr-W6/32 beads. (3) Dissociation of class I antigens from beads and analysis by the SDS-PAGE showed the presence of both free heavy and light chains of class I antigens. (4) More than 22 class I-associated peptides with a molecular weight of 700–3,000 daltons could be isolated from W6/32-loaded beads but only from lysate of HLA-positive Fen cell line. The data also showed that 1 × 106 of positive Fen cells contained about 200 µg total protein of which about 0.10 µg was class I and about 2 ng was class I-associated peptides. Conclusions: These findings demonstrated that the gene transfection approach could be used to restore missing class I antigens on an otherwise class I negative bladder tumour cell line. The results also showed the feasibility of using above techniques for isolation of HLA-associated peptides. These approaches may provide a realistic possibility for identification of putative tumour-specific peptide(s) from tumour specimens with the long-term aim to use such peptide(s) for immunotherapy in cancer patients.

Identification of tumour-specific peptide(s) hidden within the groove of human leucocyte antigens is a crucial prerequisite for peptide vaccine therapy. Conventionally, the peptide(s) are isolated by mild acid extraction (MA) technique followed by sequential ultra-filtrations. Here we describe a new approach for peptide isolation using the immunobead purification (IB-P) technique in conjunction with reverse-phase high-performance liquid chromatography. The obtained data were validated by SDS-PAGE followed by the silver staining technique. The results can be summarised as follows: (1) Comparison of class I-associated peptides isolated from a bladder cell line before and after the correction of class I antigens by gene transfection followed by IB-P technique showed the presence of peptides only from the class I-corrected cells. The data were confirmed using the silver staining technique as a way of detecting individual peptide bands. (2) Whilst peptides could be isolated by both techniques, the MA method led to the isolation of peptides from both class I-negative and class I-positive Fen cell lysates. (3) The IB-P approach could be used for isolation of class I-associated peptides from a normal kidney tissue. The data showed the high efficiency of the IB-P approach for isolation of class I-associated peptides. Unlike the MA technique, where the presence of non- class I-associated peptides was a problem, the IB-P approach isolated only peptides associated with the class I antigens. In addition, the data showed the feasibility of extracting peptides from tissue fragments by the IB-P method. The approach presented here may assist the future development of peptide vaccine therapy in urological cancers.

Objective To develop specific monoclonal antibodies (mAbs) against human germ cell tumours. Materials and methods A single-cell suspension obtained from tumour tissue fragments (consisting of both tumour and normal compartments) from a patient with seminoma was used as an immunogen. Spleen cells from immunized mice were used to develop mAbs. Tissue specificity, biochemical characteristics and competitive studies were analysed using immunocytochemical staining, dot blots and a Western blot analysis, to identify target antigen(s). Results The immunization protocol led to the development of 107 hybridomas, 90 of which were negative against the original tissue biopsies. The remaining 17 showed positivity against various tissue compartments. One selected mAb (ATC2) showed specific staining on germ cell tumours but not on normal tissues, and positive staining with some human tumour cell lines. The target antigen for ATC2 was confirmed to be placental alkaline phosphatase (PLAP) based on: Western blot analysis compared with commercially available PLAP; comparison of the data with another well-known anti-PLAP mAb (H17E2, although the two mAbs recognized different antigenic epitopes); heat resistance characteristics; high-performance liquid chromatography of the ATC2 target antigen and purified PLAP. Conclusion The selected mAb ATC2 has high specificity for human germ cell tumours, the target antigen for ATC2 being PLAP, although the antigenic epitope(s) differ from those recognized by H17E2. Thus ATC2 may be useful for monitoring serum levels of PLAP in patients with testis cancer and may be relevant for detecting cancer cells in the semen of individuals with suspected testis cancer, particularly in those with equivocal findings on ultrasonography.

The pattern of expression of cell adhesion molecules, i.e., leucocyte function associated antigen 3 (LFA-3) and intercellular adhesion molecule 1 (ICAM-1) on human bladder tumour biopsy specimens was investigated. Attempts were also made to study the pattern of induction of these molecules by established human cell lines in response to cytokines. The results indicated that 15 of 25 tumour biopsy specimens were negative for ICAM-1, and amongst the remaining 10, only 1 showed strong positivity, whilst LFA-3 was expressed in 21 of 23 cases. Unlike LFA-3, the pattern of ICAM-1 expression on established tumour cell lines was different in that there were 7 of 21 cases showing positive staining. The parallel investigation of ICAM-1 and major histocompatibility complex class II antigen expression on bladder tumours showed that in 11 of 18 cases, there was a concomitant expression or complete absence of these molecules. In the remaining 7, there were 6 cases where only class II expression was observed. Exposure of cell lines to interferons alpha or gamma had no effects on LFA-3 expression. In contrast, interferon gamma induced ICAM-1 on all the eight lines with constitutive ICAM-1 expression, whereas interferon alpha upregulated only 2 of these 8 lines. The mean +/- SD values for ICAM-1 expression on the eight inducible lines were 617 +/- 406 cpm before and 943 +/- 471 cpm (p = 0.001) after interferon gamma stimulation. The pattern of ICAM-1 inducibility of a bladder cell line Fen to interferon remained unchanged following transfection of a beta2-microglobulin gene and correction of cell surface HLA class I antigens. These results indicate that there was a significant minority of bladder tumours and tumour cell lines with abnormal cell adhesion molecule expression. In some cases, the abnormality in cell lines could not be corrected by cytokine stimulation. It is possible that these abnormalities may play a critical role in the overall tumour strategy for escape from immunological detection.

Placental alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7– 8 min (corresponding to 95 kDa molecule) and one at 12–13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.

Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

The possible role of the immune system in resisting human malignancies has long been debated. Several recent findings from animal and human studies have restimulated interest in the immune survelliance hypothesis for tumor control. These findings have been complied from various disciplines including cytokine therapy, adoptive immunotherapy, and gene therapy. Following the initial euphoria, it is now clear that immunotherapy of selected cancer cases in the early stages of tumor development may make an important contribution to tumor control, particularly in dealing with minimal residual disease after tumor debulking. This review discusses some of these issues and proposes approaches that could pave the way for better selection of the patients best suited for immunotherapy. We would argue that therapies directed at the re-expression of major histocompatibility complex (MHC) class I antigens might improve outcomes in immune-therapy–based treatments.

Objective To establish the specificity and sensitivity of alkaline phosphatase activity in testicular tumours for the early detection of testicular germ cell tumours (GCTs). Materials and methods Samples from patients with GCTs of the testis were sectioned and stained using the nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) Sigma Fast substrate technique (referred to as MEDC) and peroxidase anti-peroxidase (PAP) assay. Additionally, several cell lines from GCTs and other tumours were cultured using standard techniques and stained using both methods, and lysates from all samples were stained after gel electrophoresis. Results MEDC showed strong and specific staining of testicular tumours only. The activity on gel electrophoresis appeared as two bands, one at 75 and one at 95 kDa, which was a unique combination for testicular tumours and testis cell lines. Conclusion The MEDC method has extreme sensitivity and intensityfor testicular tumour tissue and has potential use as a powerful tool for the early detection of testicular GCTs by analysing semen samples, particularly in patients with equivocal findings on testicular ultrasonography.

The effects of the route of administration of interleukin 2 (IL-2) on the immunological parameters of patients with various malignancies were investigated. The percent mean values for IL-2 receptor (Tac) positive cells among mononuclear cells in patients receiving IL-2 subcutaneously (5 cases) or intravenously (6 cases) were 7 and 7%, respectively. The corresponding values of post-IL-2 treatment peak were 18 and 16%. Similarly, the values for class II antigen expressing cells were 12 and 16% and 25 and 26%. In no case the difference between the values for the subcutaneous or the intravenous route reached significance. Using the 51Cr release assay, the mean percent killing activity of IL-2-activated mononuclear cells against Daudi tumour target cells for the subcutaneously and the intravenously treated groups were 27 and 14% and the corresponding values for the post-IL-2 treatment peak were 59% (p > 0.05) and 69% (p > 0.05), respectively. The similar values using K562 tumour as target were 17 and 8%, and the corresponding values for post-treatment peak were 55% (p > 0.05) and 23% (p > 0.05). In all cases the cytotoxic values for the post-IL-2 treatment were significantly greater than the pre-IL-2 values. The mean (+/- SD) values for serum beta2-m levels for 6 subcutaneously and 5 intravenously IL-2-treated patients were 6.5 +/- 4.6 and 5.7 +/- 3.4 mg/l (p > 0.05). The corresponding values for post-IL-2 (15 days) were 9.1 +/- 3.4 and 9.9 +/- 5.1 mg/1, respectively. These results demonstrate that there is no significant difference in the immunological parameters in cancer patients receiving IL-2 via subcutaneous or intravenous routes and provide further support for the current trend for clinical trials to concentrate on outpatients subcutaneous administration.