Biomedical subjects

J Lawry

Publications and source records attributed to J Lawry.

At least 37 records · Page 2Linked to original sources

PubMed1995

The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression.

Tissue transglutaminase (tTgase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification of proteins via epsilon(gamma-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signaling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

Animals↗

PubMed1995

Growth stimulating antibodies in endemic goitre: a reappraisal.

OBJECTIVE: Previous studies, using a variety of methods, have reported growth-promoting immunoglobulins (IGs) in a large proportion of patients with endemic goitre. We sought to determine whether thyroid growth-promoting immunoglobulins (TGI) are present in the serum of Indian patients with endemic goitre. DESIGN: IgG was prepared by protein G-Sepharose affinity purification and added to FRTL-5 thyroid cells in the presence of suboptimal concentrations of TSH. PATIENTS: We studied 30 sequential patients with endemic goitre and 16 euthyroid controls without a goitre from the same area. MEASUREMENTS: Two assays for thyroid cell growth were used: 3H-thymidine incorporation, and flow cytometric measurement of the proportion of cells in the S phase and G2/M phase of the cell cycle. RESULTS: Both assays were shown to detect growth produced by TSH and by thyroid stimulating antibodies in IgG preparations from 3 patients with Graves' disease. There was no significant increase in either 3H-thymidine incorporation or the distribution of cells in S or G2/M phase with IgGs from endemic goitre patients, and no difference between the effects of these IgGs and those from the normal subjects. CONCLUSIONS: Thyroid growth-promoting immunoglobulins cannot be detected in Indian patients with endemic goitre.

Adolescent↗

PubMed1994

Comparison of protein synthesis profiles in chronic lymphocytic leukaemia cells and B-lymphocytes from peripheral blood, cord blood and tonsil.

2D-gel electrophoresis was used to investigate protein synthesis in leukaemic cells from a series of 15 chronic lymphocytic leukaemia (CLL) patients, and in non-malignant B-cell populations from different sources. The protein synthesis profiles of CD5+ B-cells from umbilical cord blood and from tonsil were determined, and the levels of expression of their proteins were observed to be similar to the CLL cells. The CD5- cells from cord blood resembled peripheral blood B-lymphocytes, and the protein synthesis profile of CD5- cells from tonsils was very complex. One protein was also identified which consistently appeared to be synthesised at a low level in CD5+ B-cells from tonsil but which was always more prominent in CLL cells and other non-malignant B-lymphocytes. On the basis of these data it is possible that the closest non-malignant counterpart to CLL is the CD5+ B-lymphocyte from cord blood.

Antigens, CD↗

PubMed1994

The detection of intracytoplasmic interleukin-2 in Jurkat E6.1 and human peripheral blood mononuclear cells using direct conjugate, two-colour, immunofluorescent flow cytometry.

The most commonly used approaches for the estimation of cytokine protein production involve the quantification of cytokines produced, and accumulated, in a complex body fluid or supernatant of cultured cells, by means of a bioassay or immunoassay, but these techniques do not permit an estimation of the frequency or phenotype of cytokine-producing cells. Traditional methods use immunohistochemical based techniques which can be difficult to perform and interpret, whereas flow cytometry has the advantage of objective assessment and standardisation and is less labour intensive. In this study we have established a rapid and sensitive technique for the simultaneous detection of intracellular IL-2 in conjunction with CD3. Polyclonal goat anti-IL-2 and control goat IgG were conjugated to FITC and then separated from free fluorochrome using column chromatography. PHA activated PBMC or Jurkat E6.1 cells were fixed in paraformaldehyde and permeabilised with saponin, followed by the addition of directly conjugated antibodies (FITC anti-IL-2 or PE anti-CD3) alone or in combination. Samples were then analysed using a flow cytometer and the percentage of dual labelled cells calculated. Several methods have been previously established for the detection of intracellular cytokines using flow cytometry and employing multiple layers of antibodies in the detection steps. By using direct conjugates the technique is less time consuming, requires fewer controls and can be used to examine cytokine production by identifiable cell phenotypes in a mixed cell population.

CD3 Complex↗

PubMed1994

Matrix metalloproteinase 9 expression in primary human prostatic adenocarcinoma and benign prostatic hyperplasia.

Matrix metalloproteinase (MMP) expression was investigated in patients with prostatic adenocarcinoma and benign prostatic hyperplasia (BPH). Forty-one men were studied: 26 had histologically proven prostate cancer, with 14 (54%) showing metastatic disease; 15 patients had BPH. Prostatic tissue was obtained from transurethral resection and needle core biopsies; gelatinolytic activity was determined by zymography. Seven gelatinolytic bands were detected, with molecular weights ranging from > 100 kilodalton (kDa) to 29 kDa. Nine of 14 patients (64%) with skeletal metastases had 92 kDa activity, present in only two of 12 patients (17%) with a negative bone scan, and absent in BPH. The 92 kDa gelatinolytic activity was expressed in 73% of aneuploid tumours compared with 20% of diploid tumours. A 97 kDa gelatinase was expressed in 80% of BPH samples and 23% of carcinoma patients. Enzyme bands of 72, 66 and 45 kDa were equally expressed in malignant tissue, irrespective of metastatic status, but were expressed in fewer BPH patients. The 97, 92, 66 and 45 kDa enzymes were identified as being pro-MMP-9 sequences by Western blotting, using a specific antibody directed against the pro sequence of the mature protein. MMP activity appeared to be increased in malignant prostatic tissue compared with BPH. Pro-MMP-9, in its 92 kDa form, was shown to be exclusively expressed by malignant prostatic tissue, and in particular by tumours that exhibited the aggressive and metastatic phenotype.

Adenocarcinoma↗

PubMed1993

Interleukin-6 and tumour necrosis factor alpha synergistically block S-phase cell cycle and upregulate intercellular adhesion molecule-1 expression on MCF7 breast carcinoma cells.

The effect of IL-6 and TNF alpha were studied on human MCF7 breast cancer cells. Synergistic interaction between IL-6 and TNF alpha, on the growth inhibition (50% reduction in the percentage of S-phase cells) and the upregulation of ICAM-1 expression (4 to 11-fold increase) was shown using flow cytometric methods. IL-6 and TNF alpha alone had negligible effect on the cell cycle. The individual effect of IL-6 resulted in down-regulation of ICAM-1 expression (30-35%), while TNF alpha always upregulated ICAM-1 (1.5 to 4-fold increase). The combined effect of IL-6 and TNF alpha consistently caused an increased expression of ICAM-1, which was greater than the sum of each one alone and also sustained for 72 h following cytokine withdrawal.

Breast Neoplasms↗

PubMed1993

Phenotypic and cell cycle analysis of human peripheral blood mononuclear cells activated with interleukin-2 and/or OKT3.

The poor clinical response rates of cancer patients following immunotherapy with interleukin-2 (IL-2) and other cytokines has prompted attempts to enhance the response rate by using combinations of biological response modifiers. Peripheral blood mononuclear cells (PBMC) respond to interleukin-2 and mediate non-MHC-restricted cytotoxicity and cell proliferation. The addition of anti-human CD3 monoclonal antibody OKT3 has been reported to increase cytotoxicity by increasing the number of cells generated in response to the two stimuli; however, our results could not confirm this finding. In the present study we have investigated the proliferative capacity of individual populations of PBMC responding to IL-2 and OKT3 compared to either stimulus alone, in order to identify possible reasons for the failure of OKT3 to generate enhanced cytotoxic responses. PBMC were stained with monoclonal antibodies to surface antigens and propidium iodide in order to determine the phenotype of populations of PBMC progressing through the cell cycle. OKT3 alone caused an increase in PBMC progressing through the cell cycle, and addition of recombinant human IL-2 (rhIL-2) sustained this response. A higher percentage of CD56- or CD16-positive cells responded to the rhIL-2 alone, but the addition of OKT3 lowered the percentage of this phenotype and increased the number of CD3-positive T cells, which additionally demonstrated an increased CD25 expression. Comparison of the phenotypes progressing through the cell cycle in response to OKT3 plus rhIL-2 or rhIL-2 alone showed that a higher percentage of all populations responded to OKT3 plus rhIL-2, compared to IL-2 alone. Using flow cytometric cell sorting, those populations of cells with cytolytic activity were identified. Using flow cytometry, it was also possible to show minority cell populations (i.e., those representing less than 5% of the total number of cells) responding to individual stimuli and entering the cell cycle. This technique therefore offers distinct advantages over conventional proliferation assays.

Antigens, CD↗

PubMed1993

2D-gel analysis of proteins in chronic lymphocytic leukemia cells and normal B-lymphocytes.

Protein synthesis was analysed in leukaemic cells from 10 chronic lymphocytic leukaemia (CLL) patients by 2D-gel electrophoresis of 14C-labelled proteins. There appeared to be only minor differences between each of the CLL samples, but there was evidence that the level of expression of a few of the proteins might have correlated to the stage of the disease. Comparison of the CLL samples to populations of normal B-lymphocytes demonstrated marked differences in protein synthesis between the leukaemic and non-malignant cells. We subsequently used the fluorescence activated cell sorter (FACs) to separate CD5+ from CD5- B-lymphocytes, but observed that the protein synthesis exhibited by these two populations was essentially the same, and both were very different to that observed in CLL cells. The significance of these observations with respect to the origins of CLL is discussed.

Antigens, CD↗

PubMed1993

TNF alpha protects SW742 human colon carcinoma cells against non-MHC-restricted cytolysis.

The ability of interferons (IFN alpha and IFN gamma) to protect human tumour cells from non-MHC-restricted cytotoxicity is well established. We show that in addition to rhIFN gamma, rhTNF alpha is also able to decrease the susceptibility of the human colon carcinoma cell line SW742 to non-MHC-restricted lysis by fresh and IFN alpha activated peripheral blood mononuclear cells. The observed decrease in lysis was not the result of a decrease in the rate of killing. rhTNF alpha and rhIFN gamma were unable to alter MHC class 1 expression, indicating that the protection induced was not the result of increased class 1 antigen expression; however, both cytokines enhanced ICAM-1 expression on the tumour cells. rhTNF alpha and rhIFN gamma did not alter the proliferation or cell-cycle profile of SW742 cells, indicating that the protection was not cell-cycle phase dependent and was not secondary to suppression of cell growth.

Cell Adhesion Molecules↗

PubMed1993

The identification of informative parameters in the flow cytometric analysis of breast carcinoma.

DNA ploidy and the measurement of proliferation or S-phase fraction are both of prognostic significance in breast cancer, yet clinical use is minimal in the U.K. Immunohistochemistry is, however, used to aid diagnosis, so a panel of antibodies were analysed by flow cytometry to assess their predictive value for prognosis, tumour stage and grade. Of 10 parameters tested on 202 breast tumour samples, tumour cell proliferation and DNA ploidy were the two most informative; cytokeratin staining, natural killer and B-cell infiltration also proved to be of value but there was no prognostic value in measuring tumour infiltrating monocytes, helper/suppressor T-cell ratios, tumour cell reactivity with carcinoembryonic antigen or human milk fat globulin antibodies. For each of the informative parameters, scores numerically weighted towards a poorer prognosis were derived which when combined, correlated with tumour grade, stage and prognosis. Such data interpretation is objective, and can be transposed to other human tumours.

Adolescent↗

PubMed1992

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.

Animals↗

PubMed1992

The expression of class I major histocompatibility antigens by human retinal pigment epithelium in vitro.

Since the expression of class I major histocompatibility antigens by the retinal pigment epithelium (RPE) may be important with regard to ocular immune responses, we investigated this using flow cytometry and compared the RPE cells with the colon carcinoma cell line SW742. Six cultured RPE cell lines were examined. All showed strong basal expression of class I antigen. However, this expression was not maximal, and all the RPE lines tested showed a marked capacity for up-regulation of expression by culture in the presence of interferon-alpha, interferon-gamma, or tumour necrosis factor alpha, cytokines involved in immunological responses.

Antibodies, Monoclonal↗

PubMed1992

C-myc oncogene expression in ocular melanomas.

We have investigated the expression of c-myc in 24 ocular melanomas by immunohistochemistry, using two monoclonal antibodies raised against a mid-sequence portion of the c-myc product (6E10) and against the C-terminus (9E10). The results were compared with other putative prognostic factors, including tumour size, cell type, proliferation index (determined by flow cytometry), and ploidy, as well as immunohistochemical staining for HMB-45 and S-100 antigens. Staining, often focal, for c-myc was found in both the nucleus and the cytoplasm of a proportion of the cells in most tumours studied. Total cell staining for myc protein correlated with proliferative index in diploid tumours; seven out of nine aneuploid and mixed aneuploid/diploid cells showed strong staining in at least one cellular compartment. A positive correlation with myc expression was also found for HMB-45 staining, but not for cell type or staining for S-100. The results support the hypothesis that myc protein is involved in cellular proliferation in uveal melanomas and indicate that immunohistochemistry for myc antigen may be a useful prognostic marker in these tumours.

Adult↗

PubMed1992

Circulating prostate specific antigen-positive cells correlate with metastatic prostate cancer.

Analytical flow cytometry was used to study circulating prostate specific antigen (PSA)-positive cells in 40 consecutive patients with newly diagnosed, untreated prostate cancer; 25 patients (63%) had metastatic disease confirmed by a positive bone scan. Cell suspensions were prepared for each patient from both the primary tumour and peripheral blood samples. The cells were stained with a monoclonal antibody against PSA, and analysed by flow cytometry; PSA-positive cells were sorted according to their immunofluorescence and light scatter properties. The cellular deoxyribonucleic acid (DNA) content of each specimen was also analysed to establish ploidy status. PSA-positive cells were detected in the peripheral blood of 33 patients (83%). The presence of these cells in the circulation showed a higher degree of sensitivity and specificity in predicting positive bone scans than did serum PSA levels. Circulating PSA-positive cells may represent either a subpopulation of tumour cells with distinct metastatic properties or, alternatively, host immunocytes which take up PSA in an active or passive manner.

Aged↗

PubMed1992

Evidence that interleukin-4 suppression of lymphokine-activated killer cell induction is mediated through monocytes.

Recombinant human interleukin-4 (IL-4) and transforming growth factor-beta (TGF-beta) reduce recombinant interleukin-2 (IL-2) induction of lymphokine-activated killer (LAK) cell activity from human peripheral blood mononuclear cells (PBMC). Monocytes can be removed from PBMC by adherence, leaving a peripheral blood lymphocyte population (PBL) which also responds to IL-2 to generate LAK activity. PBL generation of LAK cytotoxicity is susceptible to inhibition by TGF-beta, but not by IL-4. Readdition of purified monocytes to PBL is accompanied by return of the suppressive action of IL-4 on the generation of LAK activity. Induction of LAK cytolysis from Percoll-isolated T cells (greater than 90% CD3+) is also refractory to the inhibitory effect of IL-4. When PBMC were cultured in IL-2, with and without IL-4, subsequent sorting of CD3+ and CD3- lymphocytes by flow cytometry demonstrated that IL-4 had suppressed LAK induction in both effector populations. This suggests that, although isolated CD3+ cells are not susceptible to IL-4 suppression of IL-2 activation, they are sensitive to inhibition when part of a mixed PBMC population. Evidence is presented for the first time that this suppression is mediated via the action of IL-4 on monocytes.

Cell Adhesion↗

PubMed1990

Resurrection drug.

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History, 20th Century↗

PubMed1990

Suppression of lymphokine-activated killer (LAK) cell induction mediated by interleukin-4 and transforming growth factor-beta 1: effect of addition of exogenous tumour necrosis factor-alpha and interferon-gamma, and measurement of their endogenous production.

Recombinant human interleukin-4 (rhIL-4) and transforming growth factor-beta 1 (TGF-beta 1) suppressed the induction of lymphokine-activated killer (LAK) activity induced by recombinant human interleukin-2 (rhIL-2) in peripheral blood lymphocytes. DNA synthesis and the expression of the p55 alpha chain of the IL-2 receptor (Tac antigen) were also inhibited. The inhibitory effect was greatest when these factors were added during the first 48 h of a 4-day culture, with reduced cytolytic activity against both natural killer (NK) resistant and NK-sensitive tumour cell line targets. The suppressive action of both cytokines was accompanied by a reduction in tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) levels in lymphocyte culture supernatants. Recombinant human IFN-gamma (rhIFN-gamma), but not recombinant human TNF-alpha (rhTNF-alpha) was able to overcome the inhibitory effect of recombinant human interleukin-4 (rhIL-4) on LAK induction and DNA synthesis but not Tac antigen expression. However, cytotoxicity induced by rhIFN-gamma alone was also suppressed by rhIL-4 and TGF-beta 1, inferring that rhIFN-gamma-mediated abrogation of rhIL4 suppression was not simply a direct IL-2-independent effect on cytotoxicity. In addition, rhIL-4 did not increase TGF-beta production from rhIL-2-activated peripheral blood mononuclear cells, suggesting that rhIL-4 did not mediate reduction of rhIL-2 responses through the induction of TGF-beta release.

Cell Division↗

PubMed1990

The value of assessing cell proliferation in breast cancer.

Using three methods to measure cell proliferation, namely DNA cell cycle; anti-proliferating cell monoclonal antibody (MAb) (Ki67, P145) analysis by flow cytometry; and the histological silver (argyrophilic) staining technique to visualize nuclear-organizing regions (AgNOR), twenty-two paired samples of primary breast tumour and axillary lymph node were analysed. The results showed variable levels of correlation between the methods used for the tumour group (r = 0.915, P less than 0.001 for Ki677 versus P145; r = 0.42, P less than 0.005 for percentage S/G2/M-phase versus P145; r = 0.16, P less than 0.5 for percentage S/G2/M-phase versus AgNOR; r = 0.400, P less than 0.1 for Ki67 versus AgNOR). The lymph-node group showed slightly poorer correlations, yet involved nodes showed a consistently higher level of proliferation than non-involved nodes by all methods used. Overall, MAb binding of Ki67 or P145 was seen to be a good indicator of cycling cells, detecting G1-phase cells in addition to S/G2/M-phase cells identified by the other methods used. However, no advantage was found over the usual DNA flow cytometric analysis of cells, which had clear prognostic value. AgNOR scores were found to be able to discriminate between diploid and aneuploid; and dividing and non-dividing cells, but areas of score overlap limited the application of this technique to that of a positive discriminator only.

Aneuploidy↗