[show abstract][hide abstract] ABSTRACT: The objectives of this study were to determine the toxicity of intratumoural/intrapleural SRL172 in addition to intradermal SRL172 and standard chemotherapy (mitomycin-C, vinblastine and cisplatin) in patients with malignant mesothelioma. Patients received chemotherapy (mitomycin-C: 8 mg m(-2), vinblastine: 6 mg m(-2), cisplatin 50 mg m(-2)) on a 3-weekly basis for up to six courses. IP SRL172 injections were given 3-weekly prior to chemotherapy and escalated in groups of three patients from 1 microg to 1 mg bacilli in 10-fold increments. Patients were also given ID SRL172 at a dose of 1 mg bacilli 4-weekly. Patients were assessed for toxicity after each course of chemotherapy and for response by CT imaging. Immuno-haematological parameters were analyzed pre-treatment and 1 month after completion of treatment. There was no dose limiting toxicity with IP SRL172 although there was greater toxicity at the highest dose (n=13). There were six out of 16 partial responses (37.5%). Haemato-immunological parameters, measured in seven patients pre and post-therapy, revealed that response rate correlated with a decrease in platelet count and there was an increase in activation of natural killer cells and a decrease in the percentage of IL-4 producing T cells in all tested patients post-treatment. SRL172 can be given safely into tumour deposits and the pleural cavity in patients with malignant mesothelioma and we have established the dose for phase II testing.
British Journal of Cancer 03/2002; 86(3):336-41. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: SRL172 is a suspension of heat killed Mycobacterium vaccae, that has been found to be a potent immunological adjuvant when used with autologous cells in animal models. This is a phase II study to test the clinical activity, feasibility and safety of combining SRL172 with chemotherapy to treat patients with small cell lung cancer (SCLC).
Patients were randomized to receive chemotherapy with (n=14) or without (n=14) SRL172. The chemotherapy was either platinum-based (MVP, n=10) or anthracycline-based (ACE, n=18). SRL172 was given intradermally on day 0, weeks 4, 8 and then 3-6 monthly.
The treatment arms were well balanced for disease extent (43% with limited stage in each arm). The toxicity of chemotherapy and overall response at 12-15 weeks (57%) was the same for both treatment regimens. Median survival was 8.6 months and 12.9 for patients treated with chemotherapy alone and with the combination respectively (P=0.10). The survival trend was similar for both disease extent and chemotherapy regimen employed in favour of combination chemotherapy with SRL172.
There is a trend to improved median survival in SCLC with the combination of chemotherapy and SRL172 with no increased toxicity and irrespective of drug regimen. A phase III study examining chemotherapy in combination with SRL172 in SCLC is now underway.
[show abstract][hide abstract] ABSTRACT: It has not been assessed whether high levels of soluble interleukin 2 receptor (sIL-2R), neopterin and beta-2 microglobulin in idiopathic dilated cardiomyopathy reflect heart failure severity and/or an active autoimmune process. The aim of this study was to relate serum levels of these markers to clinical and autoimmune features.
We studied 60 patients with idiopathic dilated cardiomyopathy, 67 controls with ischemic heart failure and 34 normals.
Abnormal levels of sIL-2R, but not of neopterin and beta-2 microglobulin, were more frequent in idiopathic dilated cardiomyopathy than in ischemic patients (35% vs. 16%; P=0.02) or in normals (35% vs. 12%, P=0.01); mean sIL-2R levels were, however, similar in idiopathic dilated cardiomyopathy and ischemic heart failure (842+/-75 vs. 762+/-93 U/ml, P=NS). In idiopathic dilated cardiomyopathy abnormal levels of sIL-2R were associated with lower peak oxygen consumption (P=0.008), higher neopterin and HLA class II expression in the myocardium (P=0.02), but were unrelated to cardiac autoantibody status or titer. In addition, abnormal levels of neopterin were associated with adverse prognosis and higher beta-2 microglobulin; abnormal levels of beta-2 microglobulin with lower echocardiographic percent fractional shortening, higher sIL-2R and higher neopterin.
There is no convincing evidence that abnormal sIL-2R, neopterin and/or beta-2 microglobulin are disease-specific markers of idiopathic dilated cardiomyopathy. The lack of association with cardiac autoantibodies suggests that these abnormalities are mainly related to heart failure severity rather than autoimmune pathogenesis. In keeping with this view, high levels of sIL-2R, neopterin and/or beta-2 microglobulin identified a subset of idiopathic dilated cardiomyopathy patients with advanced disease and poor prognosis.
European Journal of Heart Failure 04/2001; 3(2):155-63. · 5.25 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed.
[show abstract][hide abstract] ABSTRACT: When lymphocytes from genetically different individuals are mixed together in tissue culture blast transformation occurs, a reaction known as the mixed lymphocyte reaction (MLR). The MLR is a clinically relevant in vitro assay where lymphocytes from one individual (effector, E) are incubated with the lymphocytes of another individual (stimulator, S) which have been previously rendered incapable of blast transformation by gamma-irradiation. We have standardised a whole blood (WB) MLR assay where the E lymphocytes were provided by 20 microl of WB and the S lymphocytes were provided by irradiated peripheral blood mononuclear cells (PBMCs) either as a mixed pool of 20 donor PBMCs or as single donor PBMC. The optimum number of S lymphocytes needed was comparatively higher than in the standard PBMC MLR: the optimum calculated E:S ratio was 1:20 compared to a E:S ratio of 1:1 or 3:2 in the standard PBMC MLR. In ten normal individuals the WB/PBMC MLR was similar to the standard PBMC/PBMC MLR. As a clinical example, the WB/PBMC MLR proliferative capacity of 13 patients with malignant mesothelioma was no different from the proliferative capacity of their age-sex matched controls. This standardised WB/PBMC MLR assay is a simple and more practical assay than the standard MLR assay and can be incorporated easily in clinical studies with biological end-points.
Journal of Immunological Methods 02/2001; 247(1-2):1-8. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein transduction can be described as the direct uptake by the cell of exogenous proteins/peptides or protein/peptide:chemical complexes, as a result of a specific property of the protein/peptide component. In this review, the three most widely studied protein transducing activities are described, with particular emphasis on the TAT protein transduction domain. Current progress in protein transduction technology suggests the potential development of a variety of molecular and cell biology tools that will enable researchers to by-pass conventional genetic routes for modulating the cells' biological activity, thus negating many of the problems associated with genetic intervention. The potential application of this class of molecule in the development of tools for the study of senescent populations is discussed.
Mechanisms of Ageing and Development 01/2001; 121(1-3):113-21. · 3.26 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mycobacterial preparations have been used with limited success against cancer apart from superficial bladder cancer. Recently, a therapeutic vaccine derived from Mycobacterium vaccae has been given to patients with prostate cancer and melanoma indicating a possible beneficial effect on disease activity in such patients. We have recently initiated a series of randomized studies to test the feasibility and toxicity of combining a preparation of heat-killed Mycobacterium vaccae (designated SRL172) with a multidrug chemotherapy regimen to treat patients with inoperable non-small cell lung cancer (NSCLC) and mesothelioma. 28 evaluable patients with previously untreated symptomatic NSCLC and mesothelioma were randomized to receive either 3 weekly intravenous combination chemotherapy alone, or chemotherapy given with monthly intra-dermal injections of SRL172. Safety and tolerability were scored by common toxicity criteria and efficacy was evaluated by survival of patients and by tumour response assessed by CT scanning. The toxicity of chemotherapy was similar in the two groups. SRL172 caused mild inflammation at the injection site. In the group of patients randomized to receive chemotherapy combined with SRL172, there was a trend towards improved response rate (54% vs. 33%) with more patients in the combined arm receiving radical surgery and radiotherapy, improved median survival (9.7 months vs. 7.5 months) and improved 1 year survival (42% vs. 18%). SRL172 appeared to improve sleep (P = 0.08) and improved appetite (P = 0.01). There was no detectable change in serum cytokine levels for gamma-interferon and TNF-alpha before and after treatment. In patients with NSCLC and mesothelioma, there may be a beneficial interaction when chemotherapy is administered in combination with SRL172. Confirmation of this effect and further investigation is underway in a randomized phase III trial and in laboratory models.
British Journal of Cancer 11/2000; 83(7):853-7. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: To investigate cancer immunotherapy using whole allogeneic (differing tissue-type) tumour cells as vaccines in the rat prostate cancer model. Materials and methods Two rat models of prostate cancer were used; MAT-LyLu tumours which grow in Copenhagen rats and PAIII tumours which grow in Lobund-Wistar rats, with crossover of the cell lines to test allogeneic vaccination. The cell lines were immunologically characterized by flow cytometry. Irradiated tumour cells were administered as subcutaneous vaccines either before tumour challenge or after tumour establishment (both subcutaneous). A preparation of heat-killed Mycobacterium vaccae bacilli (SRL172) was used as an adjuvant to increase vaccine efficiency.
Flow cytometry analysis of the cell lines showed that the PAIII cells had higher levels of major histocompatibility complex (MHC) class I and intercellular adhesion molecule (ICAM-1) expression than the MAT-LyLu cells. However, both tumour cell lines were rejected in their allogeneic hosts. Prophylactic vaccination with allogeneic MAT-LyLu cells protected against PAIII tumour challenge in Lobund-Wistar rats, with 80% of animals surviving for > 5 months, compared with 40% for animals receiving autologous cells. The immunity was prolonged, as rats were protected when rechallenged 5 months later. In Copenhagen rats allogeneic PAIII cells protected against the more aggressive MAT-LyLu tumour challenge only when the cells were combined with SRL172. Initial therapy experiments showed that vaccination with the cell lines mediated only limited tumour regression in the Lobund-Wistar rats.
The allogeneic tumour cell vaccination model described is valuable for assessing the principle and efficacy of allogeneic prostate cancer cell vaccines for clinical use.
BJU International 10/2000; 86(6):742-8. · 3.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: Natural killer (NK) cells produce multiple cytokines with potential immune regulatory roles. We standardised a whole-blood flow cytometry method to visualise intracellular cytokine production by NK cells for the study of NK cell biology and for clinical monitoring.
With a three-colour fluorescent labelling technique, specific cytokine production by NK or T cells was visualised directly in whole blood in the same sample after stimulation by phorbol 12-myristate 13-acetate (PMA) and ionomycin and by electronically gating on the CD3-ve/CD56+ve NK population or on the CD3+/CD56+ NK-T-cell population.
Detectable levels of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) but not of interleukin-2 (IL-2) or IL-4 were easily observed in NK cells. The visualisation of the cytokine production by NK cells was dependent on the addition of a Golgi transport inhibitor, Brefeldin A. Other known stimuli for NK cells (IL-2 and CD16 monoclonal antibody and incubation with K562, the NK-sensitive cell line) promoted IFN-gamma and TNF-alpha production in NK cells to a lesser extent than did PMA and ionomycin stimulation.
This whole-blood flow cytometric assay appears to be an useful and easy method to examine cytokine production by NK cells and/or by CD3+CD56+ NK-T lymphocytes in patients with relevant diseases.
[show abstract][hide abstract] ABSTRACT: HIV induces disease only following chronic activation of the immune system. Other retroviruses such as the mouse mammary tumour virus (MMTV) activate a large percentage of T cells by encoding a superantigen (SAg). To date there is no evidence that HIV encodes a SAg. An alternative way to induce pan-activation of the immune system is by allogeneic stimulation, which occurs following transplantation. Here we extend previous work which demonstrated that HIVpg120 could bind peptides in a similar manner to HLA, by demonstrating that human antigen presenting cells (APCs) expressing gp120 (but not DR1) can present a DR1-restricted peptide to induce proliferation of a DR1-restricted peptide-specific T-cell line in a similar manner to the same peptide presented by a DR1 expressing APC. Our data provide strong support for the hypothesis that the HLA-like regions of gp120 encode functional properties shared with HLA, and could explain the extraordinary clinical and immunological similarities between AIDS and chronic graft versus host disease.
[show abstract][hide abstract] ABSTRACT: To screen HIV-positive, long-term exposed seronegative and low-risk individuals for the presence of antibodies against regions of HIV-1 gp120 that share some degree of homology with HLA.
Sera were obtained from 63 HIV-1-infected subjects [52 Centers for Disease Control and Prevention (CDC) stage 2 and 11 stages 3/4], 32 HIV-exposed uninfected (HEU) subjects and from 24 low-risk HIV-1 seronegative individuals. They were tested by a peptide-based enzyme-linked immunosorbent assay (ELISA) for reactivity against peptides derived from the HIV-1 gp120 C-terminal region that contain regions of MHC sequence/structural similarity. Ten randomly selected sera from each group were also screened for anti-class I antibodies.
Thirty per cent of the long-term HIV-1-exposed seronegative individuals had antibodies against the conserved C-terminal region (C5) of HIV-1 gp120. However, sera from HEU individuals showed no reactivity against other peptides derived from the C2 region of gp120, also an HLA homologous region. Anti-C terminal gp120 antibodies were mainly of IgM subclass, although IgG-specific antibodies were also present. In addition, 70% of HEU individuals had antibodies to HLA class I molecules compared with 15% of HIV-positive patients (restricted to only those HIV-positive patients with anti C-terminal antibodies).
Our results suggest that antibody responses against the C-terminal region of HIV gp120 and HLA class I may represent markers of apparent natural protection against HIV-1 infection.
AIDS 01/2000; 13(18):2515-21. · 6.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Genetic modification of tumour cells with the GM-CSF encoding gene renders these cells more potent, as autologous tumour cell vaccine, than their wild-type counterparts. However, autologous vaccines are impractical for wide-scale clinical use and we have therefore investigated the efficacy of the GM-CSF genetic modification approach with an allogeneic whole cell tumour vaccine. In this report, we show that the allogeneic K1735-M2 (H-2k) melanoma cell vaccine induces a specific protective anti-tumour response against the syngeneic B16-F10 (H-2b) melanoma tumour in C57BL/6J mice. In vitro T cell work demonstrated that vaccination of animals with the allogeneic cell vaccine generated cytotoxic T cells specific for the autologous tumour. In vivo T cell subset depletion experiments also illustrated that this anti-tumour effect was mediated by both CD4+ve and CD8+ve T cells, suggesting that the allogeneic vaccine may operate through the 'cross-priming' phenomenon whereby tumour antigens are processed and presented to T cells by the host's own antigen presenting cells (APC). Thus, we transduced K1735-M2 cells with a GM-CSF expressing retroviral vector and showed anti-tumour activity of the GM-CSF secreting K1735-M2 cells as a therapeutic vaccine against the syngeneic B16-F10 tumour. Our data imply that GM-CSF genetically modified allogeneic whole cell tumour vaccines could be successful in the clinic. In addition, more potent combination gene therapy strategies could be tested using this therapeutic allogeneic vaccine model.
[show abstract][hide abstract] ABSTRACT: The ability of NK cells to kill tumor cells is controlled by a balance between activating and inhibitory signals transduced by distinct receptors. In murine tumor models, the costimulatory molecule B7.1 not only acts as a positive trigger for NK-mediated cytotoxicity but can also overcome negative signaling transduced by MHC class I molecules. In this study, we have evaluated the potential of human B7.1-CD28 interaction as an activating trigger for human blood NK cells. Using multiparameter flow cytometric analysis and a panel of different CD28 mAbs, we show that human peripheral blood NK cells (defined by CD56+, CD16+, and CD3- surface expression) express the CD28 costimulatory receptor, with its detection totally dependent on the mAb used. In addition, the level of CD28 varies among individuals and on different NK cell lines, irrespective of CD28 steady-state mRNA levels. By performing Ab binding studies on T cells, our data strongly suggest that binding of two of the anti-CD28 Abs (clones 9.3 and CD28.2) is to a different epitope to that recognized by clones L293 and YTH913.12, which is perhaps modified in the CD28 molecule expressed by the NK cells. We also show that B7.1 enhances the NK-mediated lysis of NK-sensitive but not of NK-resistant tumor cells and that this increased lysis is dependent on CD28-B7 interactions as shown by the ability of Abs to block this lysis. Coculture of the B7.1-positive NK-sensitive cells also led to the activation of the NK cells, as determined by the expression of CD69, CD25, and HLA class II.
The Journal of Immunology 08/1999; 163(1):62-70. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The existence of HIV positive individuals who do not appear to progress to disease, or do so only very slowly (LTNPs), strongly suggest that factors other than virus pathogenicity determine disease. The occurence of HIV infected chimpanzees that remain disease free and other African SIV infected primates where disease is apparently species specific underscores the importance of host factors [1,2]. We have examined the immune response of LTNP patients using a variety of techniques including intracellular cytokine FACscan, anchor PCR analysis of the T cell receptor and HLA typing of class II genes by DNA sequencing. Our results to date confirm that the development of disease is consistent with activation of a susceptible immune system, and that this could be due to the fact that HLA-like sequences of HIV may 'allo' activate the host immune response. In order to test this hypothesis further we have examined whether gp120 itself can bind and present specific peptides which may be capable of eliciting 'allo' activation responses in particular hosts.
[show abstract][hide abstract] ABSTRACT: To evaluate the potential of heat-killed Mycobacterium vaccae (SRL172) as a nonspecific immunostimulant and as an adjuvant to whole tumour cell vaccination in the rat model of prostate cancer.
SRL172 was used as a vaccine in the prevention and treatment of subcutaneous tumours in rats. Prevention experiments were conducted using subcutaneous MAT-LyLu tumours in Copenhagen rats, comparing vaccination with SRL172 alone, SRL172 plus autologous cells, and bacille Calmette-Guèrin (BCG) plus autologous cells before tumour implantation. Treatment experiments were conducted using subcutaneous MAT-LyLu tumours in the Copenhagen rat and subcutaneous PAIII tumours in the Lobund-Wistar rat. Tumours were induced by subcutaneous injection with tumour cells. Animals were then vaccinated with autologous cells, autologous cells plus SRL172, or SRL172 alone.
SRL172 was effective as an adjuvant to autologous whole tumour cell vaccination in the prevention of MAT-LyLu tumours and the survival benefit was equivalent to that provided when the adjuvant was live-attenuated BCG. SRL172 alone did not reduce tumour take or tumour growth in this model and neither strategy was effective in delaying the growth of established MAT-LyLu tumours. In the Lobund-Wistar rat vaccination with autologous whole tumour cells and SRL172 significantly delayed the growth of established tumours.
Mycobacterium vaccae deserves further evaluation as an adjuvant to whole tumour cell vaccination in a phase I clinical trial in patients with prostate cancer.
[show abstract][hide abstract] ABSTRACT: We have compared four cell-based tumour vaccine strategies in prevention experiments using the B16-F10 melanoma model. Two of these are thought to favour the direct antigen presentation pathway (B16-F10 expressing B7.1 and hybrids made between B16-F10 cells and macrophages) and the other two strategies are thought to act by an indirect pathway of presentation (allogeneic tumour cells and autologous tumour cells combined with a powerful adjuvant (Provax-IDEC Pharmaceuticals)). Only the two latter vaccines promoted antitumour activity, whereas the vaccines consisting of B7.1-expressing tumour cells or the hybrid vaccine failed to provide any antitumour activity. Recently human trials have commenced using transfection of the B7.1 molecule, as well as employing the hybrid technology to make tumour-B cell hybrids or tumour and dendritic cell hybrids. Our results suggest that these approaches could be disappointing in the clinics if not optimised.
[show abstract][hide abstract] ABSTRACT: The assessment of natural killer cell activity at baseline and the monitoring of this activity during treatment is important in many diseases especially in patients with cancer and AIDS. An optimised and standardised whole blood chromium release assay is described using K562 cells, the standard target erythroleukaemic cell line. The tumour cell lysis observed using whole blood is comparable to that obtained with the standard 4 h lysis assay using peripheral blood mononuclear cells as effector cells. Results with the whole blood assay are reproducible when the incubation with K562 cells is performed over a period of 18 h. The assay necessitates only 0.6 ml of blood collected in 10 IU/ml of sodium heparin as the anticoagulant. In this report, depletion experiments, also standardised using whole blood, show that the effector cells in the whole blood assay are contained within the CD56 + cell population. This assay will be of interest where the immunological status of patients with different diseases need to be frequently monitored.
Journal of Immunological Methods 09/1998; 217(1-2):177-84. · 2.23 Impact Factor