Biomedical subjects

R A Lake

Publications and source records attributed to R A Lake.

At least 19 recordsLinked to original sources

PubMed2001

Private specificities can dominate the humoral response to self-antigens in patients with cryptogenic fibrosing alveolitis.

BACKGROUND: The pathogenetic mechanisms that underlie the interstitial lung disease cryptogenic fibrosing alveolitis (CFA) may involve an immunological reaction to unidentified antigens in the lung, resulting in tissue damage. METHOD: In order to identify the range of target autoantigens, we used expression cloning, employing serum from an index patient as the probe against an expressed cDNA library that was derived from a tumour cell line. We screened over 5 x 105 recombinants and obtained sequence information on three antigens that had provoked strong responses with immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. RESULTS: All of the antigens were identifiable by comparison with sequence data from the US National Center for Biotechnology Information. Alanyl tRNA synthetase (ATS) was picked on six occasions; five of these incidences reflected independent recombination events, indicating that the library was not biased. Antibodies to ATS (anti-PL-12) represent the most common reactivity that defines the antisynthetase syndrome, which is typically expressed as polymyositis, dermatomyositis and interstitial lung disease (ILD). The index patient never showed symptoms other than those associated with alveolitis, even though sera obtained from him over a period of 2 years contained antibodies with the same specificity. Autoantibodies to ATS were never detected in serial bleeds from 11 other patients with CFA, and neither did we detect antibodies to the other two antigens identified from the serum of the index patient. CONCLUSION: The humoral response in patients with CFA can be dominated by autoantibodies with private specificities. This suggests that the antibodies are epiphenomenal and are a secondary feature of tissue damage induced by some other mechanism.

Antibody Diversity↗

PubMed2001

p53 autoantibodies in patients with malignant mesothelioma: stability through disease progression.

Malignant mesothelioma (MM) generally occurs as a pleural tumour, related to the inhalation of asbestos fibres. It is highly aggressive and largely unresponsive to treatment. The incidence of MM is particularly high in Western Australia because of the extensive blue asbestos mining operations that occurred in the north of the state until 1966. MM is unusual in that mutations in the tumour suppressor gene p53 are rarely observed, whilst over-expression of p53 protein is common. As the level of antibodies directed against p53 is thought to be of prognostic value in some cancers and as MM is known to be immunogenic, we studied a cohort of Western Australian patients to determine the prevalence of anti-p53 antibodies and their value as diagnostic markers or prognostic indicators. 6/88 (7%) of patients had high titres (>2 SD above the mean of controls) of anti-p53 antibodies. There was no correlation between antibody titre and survival. Although 3/38 (8%) of sera obtained from patients exposed to asbestos but prior to a diagnosis of MM contained antibodies, the same proportion of sera obtained from patients exposed to asbestos but who remained disease free also contained antibodies (2/40; 8%). Sera collected sequentially demonstrated a profound temporal stability in the titre of anti-p53 antibodies in patients with MM throughout the course of their illness. These results show that anti-p53 antibodies are observed only at a low frequency in the sera of MM patients and where they do occur, their elicitation is an early event that may be unrelated to antigen load. The occurrence of anti-p53 antibodies does not serve as either a useful prognostic or diagnostic indicator in MM.

Adult↗

PubMed2001

Expression and integrity of DNA topoisomerase II isoforms does not explain generic drug resistance in malignant mesothelioma.

PURPOSE: Malignant mesothelioma is a tumour that is highly resistant to a number of different chemotherapy agents, yet the mechanisms by which resistance occurs are poorly understood. The pattern of resistance is consistent with disruption of topoisomerase function or expression. Coupled with this, we have previously noted a common serological reaction to the beta isoform of topoisomerase II, suggesting that it may be aberrantly expressed in patients with mesothelioma. METHODS: We assessed the expression of topoisomerase II isoforms in sections of primary tumour. We tested a panel of five mesothelioma cell lines for sensitivity to the known topoisomerase-targeting drugs, doxorubicin and etoposide. We sequenced expressed segments of the topoisomerase genes from these cell lines that have previously been associated with drug resistance. We then investigated other potential resistance mechanisms. RESULTS: We found that the beta isoform of topoisomerase II was more frequently expressed in primary tumours. Only one of the five cell lines was highly resistant to etoposide and this cell line was found to have a point mutation in the gene for topoisomerase IIalpha. Protein levels of topoisomerase IIalpha and beta did not correlate with sensitivity to either doxorubicin nor to etoposide. Semiquantitative analysis suggested that there was marked variation in the levels of mRNA expression of MRP, gamma-GCS and MDR1. None of these findings could be associated with resistance to chemotherapy. CONCLUSION: We conclude that mutations in topoisomerase IIalpha can be associated with extreme resistance of mesothelioma to etoposide. The generic drug resistance of this tumour requires further investigation.

Adult↗

PubMed2001

Localised spontaneous regression in mesothelioma -- possible immunological mechanism.

Malignant mesothelioma (MM) is an aggressive tumor usually associated with asbestos exposure. Although it can remain stable for prolonged periods, it has not been described to spontaneously regress. MM tumors are thought to be immunogenic based both on animal studies and on the good responses in some humans treated with immunotherapy. Here we present a case of pleural MM in which a transient spontaneous regression was associated with tumor tissue infiltration with mononuclear cells and serological evidence of anti-MM reactivity. The patient's tumor eventually progressed and with this progression there was evidence of loss of serological reactivity to some, but not all, of her MM antigens. The patient survived for 20 months and, in contrast to her initial biopsy, no significant lymphoid infiltrate was detected in her MM tissue at post mortem examination.

Antibodies, Neoplasm↗

PubMed2001

Lack of ignorance to tumor antigens: evaluation using nominal antigen transfection and T-cell receptor transgenic lymphocytes in Lyons-Parish analysis--implications for tumor tolerance.

A substantial body of literature has described weak antitumor CTL responses in tumor-bearing hosts, and a number of authors have suggested that tumor tissue in some way sequesters antigen from the immune system, a failure of the tumor-specific immune response largely attributable to "ignorance." To evaluate this in a tumor model, we stably transfected murine tumor cell lines with genes coding for the nominal antigens influenza hemagglutinin (HA) or ovalbumin (OVA) and adoptively transferred HA- or OVA-specific T-cell receptor-transgenic, CD8-positive T cells into mice-bearing these tumors. Tumor antigen cross-presentation within draining lymph nodes (LNs) was then examined using Lyons-Parish analysis, detection of a proliferative response of 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled CD8 T cells from T-cell receptor mice using flow cytometric analysis. Our studies demonstrate clearly that tumor antigens are constitutively presented in LNs draining tumors and can stimulate a T-cell proliferative response. This lack of ignorance was not simply attributable to the model chosen, because it was seen with three different cell lines, two different antigens, and two different mouse strains. Furthermore, it occurred regardless of whether these tumor antigens were expressed as cytoplasmic, transmembrane, or secreted proteins. When tumor antigens were present in low concentrations, antigen cross-presentation was not absent but simply delayed. Interestingly, tumor antigen cross-presentation remained localized to the LNs draining the tumor throughout the period of tumor growth. Curiously, in animals where tumors failed to grow, evidence of continued cross-presentation of the tumor antigen was seen up to 6 months after tumor inoculation. These data suggest that ignorance is not an explanation for the failure of the host immune system to respond to tumor antigens.

Animals↗

PubMed2000

Tumor-specific CD4+ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity.

A number of tumor studies have indicated a link between CD4 help and the magnitude and persistence of CTL activity; however, the mechanisms underlying this have been largely unclear. To evaluate and determine the mechanisms by which CD4(+) T cells synergize with CD8(+) T cells to prevent tumor growth, we used the novel technique of monitoring in vivo CTL by labeling target cells with CFSE. This approach was supported by the direct visualization of CTL using peptide-MHC tetramers to follow tumor-specific T cells. The data presented demonstrate that while cotransfer of Ag-specific CD4(+) T cells was not required for the generation of CTLs, because adoptive transfer of CD8(+) T cells alone was sufficient, CD4(+) T cells were required for the maintenance of CD8(+) T cell numbers. Our data suggest that there is a correlation among the number of CD8(+) T cells, in vivo CTL function, and IFN-gamma production, with no evidence of a partial or nonresponsive phenotype among tetramer-positive cells. We also show that CD4(+) T cells are required for CD8(+) T cell infiltration of the tumor.

Adoptive Transfer↗

PubMed2000

Structure of the Mesothelin/MPF gene and characterization of its promoter.

Mesothelin is a cell-surface antigen of unknown function that is expressed in a highly tissue-specific manner. Expression of the protein is limited to mesothelium, the tissue forming the pleural, pericardial, and peritoneal membranes. The purpose of this study was to characterize the gene and identify elements responsible for its transcription. The gene contains an 1884-bp open reading frame encoded by 15 exons occupying 8 kb of human chromosome 16. An 1850-bp region of genomic DNA at the 5' end of the gene encompassing the proposed transcriptional start site was cloned. This region lacks a TATA box and other regulatory elements such as SP1 sites, which are commonly found in promoters. Transient transfection analyses demonstrated that mesothelium-specific control elements are present within the 1.85-kb region. Minimal constitutive promoter elements were localized to a 317-bp region. Tissue-specific enhancer elements upstream of the minimal promoter were found to activate transcription from the homologous and a heterologous promoter in a position- and orientation-independent manner.

Blotting, Northern↗

PubMed2000

New chemotherapeutics in malignant mesothelioma: effects on cell growth and IL-6 production.

PURPOSE: The benefits of chemotherapy can be assessed in terms of tumour shrinkage, prolongation of life or simply palliation of symptoms. In the study reported here, in vitro correlates of these parameters were sought as a rational guide to the choice of newer agents in the clinic. METHODS: The cytotoxicity and effects on IL-6 production of ten chemotherapy agents representing four different classes of drugs were tested against a panel of five mesothelioma cell lines. RESULTS: The mesothelioma cells were more sensitive to the action of irinotecan (and its active metabolite SN38) and gemcitabine than the control cell lines. Gemcitabine and to a lesser extent irinotecan inhibited the secretion of the proinflammatory cytokine IL-6 at concentrations of each drug that produced only small decreases in cell viability. This effect was not seen in cells treated with docetaxel or vindesine. Higher doses of gemcitabine and irinotecan caused a surge in IL-6 release and this was not due to release of intracellular stores of IL-6 through lysis of the cells. CONCLUSIONS: These results suggest that irinotecan and gemcitabine are not only more likely to be active against mesothelioma than other new chemotherapy agents but may also produce a palliative effect in nonresponders to these agents by decreasing the secretion of IL-6.

Antimetabolites, Antineoplastic↗

PubMed2000

Replication-restricted vaccinia as a cytokine gene therapy vector in cancer: persistent transgene expression despite antibody generation.

BACKGROUND: As antitumoral immunity requires the generation of local immunity directed against tissue proteins, we attempted to recreate within tumors the same environment found within tissues affected by autoimmune diseases (i.e., prolonged cytokine expression). Vaccinia virus (VV) has not been widely used as a cytokine gene therapy vector because of presumed high immunogenicity that would likely make repeated injections impossible; therefore, we modified it by inserting the cytokine gene into the thymidine kinase region, rendering it replication-restricted. The cytokine chosen was human interleukin-2 (IL-2); a molecule with powerful antitumoral effects. METHODS: Six patients with the treatment-resistant tumor malignant mesothelioma received intratumoral (i.t.) VV-IL-2 therapy for 12 weeks by injection of 10(7) plaque-forming units of VV-IL-2 per dose. Serial tumor biopsies, sputum, urine, and blood samples were tested for VV-IL-2 mRNA expression; VV culture and T-cell infiltrates were evaluated by immunohistochemistry. Patients and contacts of patients were monitored for changes in VV immunoglobulin G (IgG) levels and clinical evidence of VV infection. RESULTS: VV-IL-2 was not excreted and was only cultured in one patient from tumor biopsies. A T-cell infiltrate was detected in 50% of tumor biopsies. VV-IL-2 mRNA expression was highest on days 1-3 postinjection and was detected for up to 3 weeks after each injection even though VV IgG levels rose in all patients. No significant toxicities, infection of patient contacts, or tumor regressions were observed. CONCLUSIONS: I.t. VV-IL-2 administration is safe, is associated with minimal toxicity, and results in i.t. expression of VV-IL-2 for up to 3 weeks postinjection regardless of the level of anti-VV IgG titers generated. This suggests that VV may be a good vector for repeated cytokine gene therapy of solid human cancer.

Adult↗

PubMed2000

Partial T cell activation with an altered superantigenic ligand.

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.

Binding Sites↗

PubMed2000

Altered superantigenic ligands demonstrate the quantitative nature of T-cell activation.

In a recent study, a superantigen mutated in the TCR binding site (staphylococcal enterotoxin B (SEB)delta61Y) was described, which behaved as a partial agonist for a Vbeta17-expressing T-cell clone. Evidence is now presented to demonstrate that there is distinct heterogeneity in the response of primary T cells to this protein. Some Vbeta17 T cells responded to SEBdelta61Y by modulating surface receptor expression consistent with activation, and by proliferating. Other Vbeta17 T cells did not proliferate, nor did they display a receptor expression phenotype consistent with activation. However, when repeatedly exposed to the altered superantigen, some of these non-responders entered cell cycle. This pattern of responses was not recapitulated by providing additional costimulation via CD28, although such treatment did induce some of the 'unresponsive' Vbeta17 T cells to upregulate the IL-2 receptor, indicative of partial activation. It was also found that the heterogeneous pattern could be replicated using very low doses of native SEB. The data are discussed in the context of models of T-cell activation in which differences in TCR ligand affinity and dose determine qualitatively different response phenotypes.

CD28 Antigens↗

PubMed2000

Simian virus (SV) 40 like sequences in cell lines and tumour biopsies from Australian malignant mesotheliomas.

BACKGROUND: Simian virus (SV) 40 sequences have been found in some, but not all studies of mesotheliomas. This virus is known to cause tumours in rodents but its role in human oncogenesis remains controversial. AIMS: The aim of this study therefore was to determine whether SV40 is associated with the development of mesotheliomas in Australia. The absence of the virus or its gene products in tissue derived from mesotheliomas would detract from this possibility. METHODS: We used polymerase chain reaction from three pairs of primers to amplify different regions of the large T antigen from DNA from cell lines and cDNA from both cell lines and an independent set of tumour biopsies from patients with mesothelioma. RESULTS: We examined five human mesothelioma cell lines that were established in our laboratories. In addition, we examined several tumour biopsies from seven different patients. SV40 like sequences were present in all the cell lines and in at least one sample from each of the patients examined. CONCLUSIONS: The large T antigen of SV40 or an SV40 like virus is expressed in Australian mesotheliomas and therefore could be aetiologically-associated with tumourigenesis. Alternatively, these sequences could be expressed subsequent to the development of the disease.

Antigens, Viral, Tumor↗

PubMed2000

Serologic responses in patients with malignant mesothelioma: evidence for both public and private specificities.

Malignant mesothelioma (MM) is a pulmonary malignancy that appears to be immunogenic based on a large number of studies in both animals and humans. This notion is supported by our recent demonstration using Western blot analysis of immunoglobulin G antibodies reactive with a variety of autoantigens in many patients with MM. In view of the enormous potential of such antigens in early diagnosis, immunotherapy, and vaccination of at-risk individuals, it was essential to identify these antigens. We therefore applied the SEREX technique (serologic identification by recombinant expression cloning), using a serum pool from six patients as the probe against an expressed complementary DNA library derived from a cloned MM cell line. We screened over one million recombinants and obtained sequence information on eight antigens that had provoked immunoglobulin heavy chain class switching, presumably as a consequence of T-cell recognition. Six of these antigens were identifiable (U2AF[65], Siah binding protein, topoisomerase IIbeta, ZFM1, mIre1, and pendulin), and of the others, one was found as a single EST from a myotube library (Jemm-1); the other (Jemm-2) was not represented in any EST database even as a weak homolog. Consistent with our previous findings, each of the characterizable antigens would be expected to be associated with the cell nucleus. Each of the autoantibody specificities was uniquely associated with a single patient with the exception of antibodies to TOPIIbeta and U2AF(65). We found 13 of 14 (93%) patients with MM had antibodies to TOPIIbeta and two of 14 (14%) patients had antibodies to U2AF(65). The number of serum reactivities, taken as a measure of the complexity of the immune response, correlates with patient survival and with an index of systemic inflammation. These data suggest that a broader range of serologic reactivities reflects a more active host response to the presence of tumor.

Animals↗

PubMed1999

Tumor antigens are constitutively presented in the draining lymph nodes.

Tumor growth is rarely associated with a strong specific CTL response, suggesting that the immune system is ignorant of the presence of tumor because the Ags are not readily available to or are sequestered from potential effector cells. We studied the in vivo activation of naive TCR transgenic hemagglutinin (HA)-specific CD8+ T cells adoptively transferred into mice bearing HA-expressing tumor using 5,6-carboxy-succinimidyl-fluorescein-ester labeling, which allows the identification of proliferating HA-specific T cells. We demonstrate that tumor Ags are constitutively presented in the lymph nodes draining tumors and are powerfully mitogenic for responding T cells despite the absence of anti-tumor CTL responses. Importantly, this proliferative signal occurs throughout tumor growth and is still detectable 6 mo after tumor inoculation when tumor is not palpable. These results provide the first evidence that there is constitutive presentation of tumor Ags in draining lymph nodes.

Animals↗

PubMed1999

T-cell receptor transgenic analysis of tumor-specific CD8 and CD4 responses in the eradication of solid tumors.

The role of tumor-specific CD8 and CD4 lymphocytes in rejecting solid tumors has been difficult to determine because of the lack of models in which tumor antigen, specific CD8 cells, and specific CD4 cells can be monitored and controlled. To investigate the minimal components required for the induction and maintenance of CTL activity sufficient to reject a solid tumor in vivo, we transfected the influenza hemagglutinin (HA) gene into a nonimmunogenic class I+/class II- murine malignant mesothelioma (MM) tumor line to generate an endogenous tumor antigen and used TCR transgenic mice with class I- or class II-restricted specificities for HA as sources of naive, tumor-specific T cells. The data show that the presence of a strong tumor antigen is not in itself sufficient to induce an effective CTL response, nor does the presence of a high frequency of precursor cells guarantee tumor rejection. We also show that tumor-specific CD4 cells, when CTL numbers are suboptimal, greatly enhance the eradication of tumor, confirming the importance of antigen-presenting cell presentation of tumor antigens to class II-restricted cells. These data confirm that T-cell receptor transgenic cells, combined with nominal tumor antigen transfection, represent powerful tools to analyze tumor-specific T-cell responses.

Animals↗

PubMed1999

MHC multimerization, antigen expression and the induction of APC amnesia in the developing immune response.

Class II multimer formation is an important event in immune recognition. Not only is multimerization a prerequisite for T cell activation, but it is a signal to APC. In the present article, we propose that multimerization can result in the specific removal of ligand complexes from the cell surface of the APC, an event which may influence the overall pattern of T cell reactivity.

Animals↗

PubMed1999

Cross-presentation of tumour antigens: evaluation of threshold, duration, distribution and regulation.

The development of technology to measure antigen presentation in the secondary lymphoid system has provided the opportunity of analysing components of the host antitumour immune response that have, until now, been unavailable for study. In particular, this technology has enabled us to evaluate threshold levels of tumour antigen required for cross-presentation in draining lymph nodes, the duration of this antigen presentation and processes that regulate tumour antigen presentation. Thus, we have been able to dissect out the relationship between antigen presentation and the resultant development of effector function in class I-restricted T cells, as well as the role of regulatory CD4 cells. We have also used this technology to evaluate the effects of antitumour therapy on local antigen cross-presentation.

Adoptive Transfer↗

PubMed1998

Sera from patients with malignant mesothelioma can contain autoantibodies.

Malignant mesothelioma (MM) is resistant to all conventional forms of therapy though there is considerable evidence from clinical trials and animal models of the disease that an immune response can be elicited to the tumour. In order to define those target antigens expressed by MM cells which might provide a focus for an effective immune response we tested patients' sera for the presence of MM autoantibodies by Western blot analysis. Eight of 29 (28%) patients with MM had serum antibodies of the IgG class in high titre and each antiserum recognised different protein antigens. In those individuals where sequential samples were available, the antibody titre increased with the progression of the disease though the number of target antigens remained constant. Sera from the eight patients were studied further: six of the antigen complexes were expressed at least partially in the nucleus; two showed some specificity for the tumour in that they discriminated antigens that were highly expressed in all human MM cell lines, but were not expressed in a human SV40 transformed mesothelial line; four of the antisera recognised a homologue in mouse tissue and each of these had a different pattern of expression. Collectively, these antisera define a subset of nuclear autoantigens that are over-expressed in dividing cells.

Adult↗