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Biomedical subjects

A M Nouri

Publications and source records attributed to A M Nouri.

At least 19 recordsLinked to original sources
PubMed2001

Combination of HPLC and solid-phase binding assay for isolation and purification of MHC class I and associated peptides using a bladder tumour cell line.

The major histocompatibility complex (MHC) class I molecules present processed self and non-self peptides to T lymphocytes. Given that the class I peptide complex plays a critical role in cell-mediated immunity, it is important to identify the nature of class I-associated peptides unique to malignant cells as a prelude to the development of vaccines. The aim of this study was to combine immuno-bead purification (using anti-class I antibody W6/32) technique, sequential ultra-filtration and high performance liquid chromatography (HPLC) to isolate class I antigens and associated peptides from an in-house established bladder tumour cell line (Fen) whose missing class I antigens had been restored by beta2-microglobulin (beta2-m) gene transfaction. The results were as follows: (a) class I antigens could be separated from tumour cell lysate but only from the class I positive Fen cells; (b) treatment of CNBr-W6/32 beads pre-exposed to class I positive Fen lysate and eluted with dissociation agent (mild acid) resulted in the release of more than 20 peptides at an approximate molecular weight of between 700 and 3000 Da based on SDS-PAGE and silver staining analysis; (c) purified and eluted peptides from class I antigens showed distinct peaks when analysed by HPLC. The data presented in this investigation demonstrated the feasibility of isolating class I antigens and associated peptides from a bladder tumour cell line. The extension of these approaches to isolate peptides from tissue tumour biopsies may help the future of vaccine therapy in cancer patients.

Chromatography, High Pressure Liquid↗
PubMed2000

The combined use of high performance liquid chromatography and immuno-biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins.

HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

Alkaline Phosphatase↗
PubMed2000

Profile of p53 expression in bladder and oral tumours. Effects of in vitro manipulations of p53 on the behaviour of established human tumour cell lines.

In this investigation the profile of p53 and epidermal growth factor receptor (EGFR) expression in tumour tissue biopsies of transitional cell carcinoma of bladder (TCC) and of oral-pharyngeal carcinoma (OP) were compared using an immunocytochemical staining method. In addition, various techniques including sodium dodecyl sulphate-polyacrylamide gel elecrophoresis (SDS-PAGE), colorimetric assay and gene transfection were used to investigate the influence of p53 on the behaviour of human tumour cell lines in vitro. The results showed that: (a) p53 was detectable in more than 45% of cases in both tumour types, although the profile and intensity of expression differed. (b) Concomitant strong expression of EGFR and p53 for TCC and OP was 21% and 38% (P>0.05%), respectively. (c) Treatment of tumour cells by either gamma radiation or by cisplatin resulted in the induction of p53 independent of the origin of the tumour. (d) Susceptibility of two cell lines, one with and one without constitutive expression of p53 showed that the expressing cells were more sensitive to gamma radiation (the percentage inhibition at 250 cGy was 57% versus -15%, P<0.01), and also cisplatin (the percentage inhibition at 1 microgram/ml was 71.0+/-6.0 versus 2.6+/-7.0, P<0.001). (e) Transfection of wild-type TP53 gene into a bladder tumour cell line resulted in a rapid cell apoptosis (by as much as 90%) whereas cells receiving mutated TP53 survived. A similar frequency of TP53 mutation in TCCs and OPs was observed. In addition, the pattern of p53 expression within the squamous type of TCC was similar to that in OPs. If the data from the in vitro studies could be translated into an in vivo setting, one could envisage a situation where the introduction of wild-type TP53 gene by gene transfection into tumour cells (independent of their TP53 gene mutational status), would prove to be beneficial. If the cellular TP53 gene is mutated, then an introduction of the normal TP53 gene would induce cells to undergo apoptosis. Alternatively, if TP53 is wild-type, then the increased levels of p53 expression would enable the cells to become more susceptible to DNA damaging treatments such as cisplatin or gamma radiation.

Carcinoma, Transitional Cell↗
PubMed2000

A new highly specific monoclonal antibody against placental alkaline phosphatase: a potential marker for the early detection of testis tumour.

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.

Alkaline Phosphatase↗
PubMed2000

Profile of epidermal growth factor receptor (EGFr) expression in human malignancies: effects of exposure to EGF and its biological influence on established human tumour cell lines.

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

Alkaline Phosphatase↗
PubMed1999

Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression.

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.

Alkaline Phosphatase↗
PubMed1999

Standardization and potential use of HPLC for detection of cellular placental alkaline phosphatase using established tumour cell lines and fresh tumour biopsies.

Placement 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.

Alkaline Phosphatase↗
PubMed1999

Characteristics of visitors to traditional healers in central Sudan.

Traditional healing is widespread in Sudan and traditional healers are well respected by the community. This study aimed to assess the characteristics of visitors attending traditional healers, the reasons for visits, the frequency of visits, satisfaction with visits and advantages and disadvantages of visits. The results showed that children under ten years did not take part in visits; most of the visitors were between 21 and 40 years (61%) and were women (62%). Visitors were less educated compared to the general population in the area. The main reasons given for attending traditional healers were treatment (60%) and blessing (26%). Visitors did not mention any disadvantages to visiting traditional healers.

Adult↗
PubMed1998

Relevance of the immune system in human urological malignancies: prospective for future clinical treatments.

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 surveillance 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.

Cell Line↗
PubMed1998

Comparative expression of major histocompatibility complex (MHC) antigens on CD5+ and CD5- B cells in patients with chronic lymphocytic leukaemia (CLL)

The aim of this study was to investigate the expression of major histocompatibility complex (MHC) antigens on CD5+ and CD5- B cells of 13 patients with chronic lymphocytic leukaemia (CLL). This was carried out using a series of monoclonal antibodies (MAbs) against polymorphic and monomorphic class I and class II antigens, as well as to the transferrin receptor and assessed by flow cytometry and direct and indirect immunofluorescence. The expression of these molecules was assessed as mean fluorescent intensity (MFI). The results showed that cells from all 13 individuals expressed monomorphic class I antigens. The number of cases expressing polymorphic HLA-Bw6, -Bw4, -B7, -B27 and -A2 class I antigens on CD5- B cells was 11 (85%), 6(46%), 2(15%), 1(8%), 3 (23%), respectively, which was consistent with the expected population frequency distributions of these antigens. For each of the class I antigens on CD5+ and CD5- B cells, the ratio of the MFI was greater than 1 in 12 of 13 cases. For the transferrin receptor (CD71), this ratio was also almost always greater than 1. These results indicate that, unlike solid tumours where the loss or abnormal expression of class I and II antigens is a frequent event, the expression of class I antigens in CLL patients seems to be normal. This indicates that the loss of these antigens cannot provide the leukaemic cells with a selective advantage to escape immunological detection.

Antigens, Neoplasm↗
PubMed1998

Malignancy associated papillomaviruses and morphology of human bladder cancer.

Animal studies in rabbit and cattle have clearly demonstrated the contribution of host genetics, chemical carcinogens and immunosuppression to the conversion of papillomavirus induced benign regressing warts into malignant cancers. More significant is the role of vaccination both with whole tumour cell suspensions with whole virus and viral proteins, particularly L2 molecules, in causing progressing warts to regress. Early results in small scale studies of HPV16 E6/E7 vaccine in patients with cervical cancer have provided evidence that tumour regression can be induced in human papillomavirus induced tumours. These observations provided added impetus for more research to firm up the increasing, but still principally anecdotal, evidence that papillomaviruses may be involved in the pathogenesis of bladder cancer. Studies of carcinomas arising in cattle after BBV 4 infection show absence of fully infectious virus in the majority of tumours, though the tumours have persistent E7, E8 and LCR sequences. As this is all that is required for transformation, it may require in vitro molecular studies in human bladder cancer screening for such elements before final proof of involvement is confirmed. However, even before this is achieved, given the success in animal models of whole tumour cell vaccines, serious thought should be given to how to develop protocols for study of crude tumour cell vaccines in vivo. Such studies would need in vitro assays to seek evidence for specific antitumour immunity, focusing on studies of tumour infiltrating lymphocytes and their T cell receptor polymorphisms.

Animals↗
PubMed1998

Clonal development of bladder cancer and its relevance to the clinical potential of HLA antigen and TP53 based gene therapy.

In the last decade there has been major progress in understanding the multiple steps involved in cancer cells developing their malignant potential. Study of bladder cancer has provided important information during this period and helped to identify HLA class I and wild type normal TP53 as potential probes for gene therapy studies. Given the magnitude of genetic damage that is associated with the clonal development of bladder cancer, and particularly the number of cellular mechanisms that have been highjacked in the development of terminal metastatic disease, it is obviously highly unlikely that a single gene therapy involving HLA class I alone would work in terminal metastatic disease. However, it seems possible that HLA-B7 treatment of patients with bladder cancer who are candidates for salvage cystectomy would benefit such patients. If it were to work, it could provide a whole new approach to managing superficial tumours. However for patients with extensive metastatic disease, progress could come from investing more effort in uncoverinlg the genetic basis of the chemosensitivity of germ cell tumours and understanding the basis of the normal checkpoints of meiosis and the role of TP53. This could provide a completely new approach to gene therapy that might be exploited even in patients with advanced metastatic disease. Such a tetraploidal construct combined with allogeneic HLA class I and incorporated into a low pathogenic lytic viral construct to induce oncolysis with some form of tissue promoter to focus the cell types in which the genes will be activated could provide ideal effective gene therapy.

Carcinoma, Renal Cell↗
PubMed1997

A new approach using tissue alkaline phosphatase activity to identify early testicular cancer.

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 antiperoxidase (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 intensity for 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.

Alkaline Phosphatase↗
PubMed1996

Comparison of immunological changes between subcutaneous and intravenous route of administration of IL-2 in cancer patients.

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.

Biomarkers↗
PubMed1996

Defective expression of adhesion molecules on human bladder tumour and human tumour cell lines.

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.

Biopsy↗
PubMed1996

Accurate quantitative analysis of cellular proteins using a computerised scanning-aided dot blot technique.

The aim of this study was to use a dot blot technique (DB) aided by a computerised scanning programme (CSDBT) to assess expression of cellular proteins in various tumour cell lines. Two groups of proteins, i.e. major histocompatibility complex (MHC) Class I and cell adhesion molecules (e.g. ICAM-1) were investigated. The constitutive expression of Class I antigens was measured and found to vary for different tumour lines. Exposure of SKV14 (SV40 transformed epithelial cells) and 5637 lines (bladder) to interferons (IFN) alpha or gamma resulted in upregulation of Class I antigens. IFN gamma, but not IFN alpha, induced ICAM-1 on all the lines studied. Thus, the stimulatory indices (SI) for Class I antigens in the case of SKV14, J82 (bladder) and 5637 lines after IFN gamma and IFN alpha stimulation were 2.02, 1.77, 4.68 and 1.65, 1.36, 2.08 respectively. The corresponding values for ICAM-1 were 1.65, 1.87, 3.43 and 1.75, 1.0, 0.96. In all the cases, the P values (using a paired t-test), except ICAM-1 for IFN alpha-stimulated J82 cells, were below 0.05. Following exposure of cells to combinations of drugs and IFN alpha, the percentage inhibition for Class I and ICAM-1 in the case of SKV14 and 5367 lines in the presence of cycloheximide (10 micrograms/mL) were 82%, 85% and 57%, 45% respectively. The corresponding values for indomethacin (10 micrograms/mL) were -23%, -52% and -20%, -24%. In all cases, the P values (using a paired t-test) were below 0.05. To our knowledge this is the first report describing a computerised scanning programme for assessing the presence of cellular proteins. The results were consistent with those obtained by radiobinding and immunocytochemistry. This simple and sensitive approach highlighted two important issues: (a) that it can be used to detect minor changes, under various conditions, of cellular proteins whether cytoplasmic or otherwise; and (b) that the accuracy of the measurements was enhanced by the use of computer scanning.

Biomarkers, Tumor↗