Simian virus 40 detection in human mesothelioma: reliability and significance of the available molecular evidence.

Simian virus 40 was discovered as a contaminant of early poliovirus vaccines that were inadvertently administered to millions of people in Europe and the United States between 1955 and 1963. Although SV40 was proven to be oncogenic in rodents and capable of transforming human and animal cells in vitro, its role in human cancer could not be proven epidemiologically. The matter was forgotten until 1993 when SV40 was accidentally found to cause mesotheliomas in hamsters injected intra-cardially. Subsequently, DNA sequences associated with its powerful oncogenic principal, the large T antigen, were found with high frequency in human pleural mesothelioma using the polymerase chain reaction (PCR). Since then many laboratories have confirmed the human findings. However, a few laboratories have failed to reproduce these data and the authors of the studies have claimed that the detection of SV40 DNA may simply represent PCR contamination artefacts. The controversy raised by this viewpoint is reviewed in this article together with a critical appraisal of the reliability of the molecular techniques used to detect SV40 DNA, in order to evaluate the potential aetiopathogenic role of SV40 in human mesothelioma.

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure. A 10 year review of its principle reagents and applications.

Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labelled primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or anti-DNP linked marker enzyme. In this article the development of these reagents and various modifications of the basic technique are reviewed in conjunction with the special applications accruing from their use.

Immunoreactive p53 and metallothionein expression in duct carcinoma in situ of the breast. No correlation.

Immunocytochemically detectable MT and p53 have been found more commonly in comedo DCIS of the breast with high-grade cytology. The aim of this study is to confirm these findings and to investigate the relationship between MT and p53 in a single large series of cases of DCIS of the breast. To this end, 127 cases of DCIS were classified histologically according to architecture, cytonuclear differentiation (grade), presence and extent of intraduct necrosis, and using the Van Nuys system. Sections were immunostained for p53 and MT (E9) using established techniques, and the extent and intensity of staining were assessed semi-quantitively. The results confirmed that there was generally more MT and p53 positivity in poorly differentiated (grade 3) DCIS with extensive necrosis and that MT expression was greater in grade 2 lesions than p53 expression. However, overall there was no statistically significant correlation between p53 and MT staining. The results indicate that MT and p53 overexpression may arise from independent mechanisms in early breast neoplasia.

Ampligen: a potential toll-like 3 receptor adjuvant for immunotherapy of cancer.

Therapeutic vaccination against cancer-associated antigens represents an attractive option for cancer therapy in view of its potential efficacy, the possibility of long-lasting immunity against the cancer, and safety profile. Cancer patients are however usually immunosuppressed and most cancer-associated antigens are 'self-antigens', making it a significant challenge to vaccinate patients against a cancer-associated antigen. Various immunostimulant adjuvants are therefore under investigation in an effort to boost the immune system to overcome the tolerance to tumour associated self-antigens and the ambient immunosuppressory influence of cytokines and regulatory T-cells (Tregs). Many adjuvants appear to be specific ligands for toll-like receptors (TLR) which are potent activators of innate immune responses [Kwissa M, Kasturi SP, Pulendran B. Expert Rev Vaccines. The Science of Adjuvants 2007 6(October(5)):673-84] [1], activating dendritic cell (DC) maturation and inflammatory cytokine secretion, inducing an increase in the cross talk between the innate and adaptive immune systems, and thereby driving the expansion of CTLs that can destroy cancer cells. However, recently some TLR agonists such as CpG have been shown to generate parallel immunosuppressive and inflammatory responses in innate immune cells capable of induction and expansion of Tregs [Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008 27(January 7(2)):168-80 [2]; Huang B, Zhao J, Unkeless JC, Feng ZH, Xiong H TLR signaling by tumor and immune cells: a double-edged sword. Oncogene 2008;27(2):218-24] [3]. In this context it is noteworthy that poly(I:C), a synthetic double-stranded RNA polymer and a TLR3 agonist, has been shown in mouse models to be capable of enhancing the T cell response to nonimmunogenic self-antigen linked dendritic cell based vaccine strategy, and stimulating diabetic insulitis in the presence of Tregs [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65-72] [4]. Ampligen poly(I:C(12)U) is a GMP-grade synthetic analogue of poly(I:C) and therefore suitable for human use. Its effects in a preclinical setting have been recently tested to be similar to poly(I:C) [Hornum L, Lundsgaard D, Markholst H. PolyI:C induction of diabetes is controlled by Iddm4 in rats with a full regulatory T cell pool. Ann N Y Acad Sci 2007;1110:65-72]. In particular, it potently induces in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. In addition human monocyte derived DC primed with tumour lysate and matured with Ampligen are capable of generating Th1 specific anti-cancer responses in peripheral blood T-cells derived from cancer patients in the presence of ascites medium containing immunosuppressory cytokines. In this review the key findings are summarised and discussed with a view to offering a rationale for the use of Ampligen as a potentially safe adjuvant capable of overcoming the tumour-related immune tolerance mechanisms in a clinical setting.

A clinical grade poly I:C-analogue (Ampligen) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro.

Maturation of dendritic cells (DC) can be triggered in vitro by inflammatory cytokines or Toll-like receptor (TLR) ligands such as CpG or polyI:C. Corresponding, well-characterized agents which can be applied in clinical settings are sparse. We have evaluated a clinical grade, non-toxic analogue of polyI:C, poly(I:C12U) (Ampligen), as a potential adjuvant for cancer immunotherapy, for its ability to drive maturation of human myeloid DC. Our results provide evidence that poly(I:C12U) is effective in inducing optimal phenotypic (elevated levels of MHC-Class I/Class II, CD83, CCR7, CD86 and CD40 molecules) and functional maturation of human DC in vitro, capable of promoting the production of the inflammatory (Th1-type) cytokine IL-12, with significantly lower levels of IL-10 production, compared to that induced by the parent compound polyI:C. Importantly, poly(I:C12U) has a comparable effect on the maturation and function of DC derived either from healthy donors or cancer patients indicating that it is able to overcome any immune suppressive factors associated with the tumour bearing state. These characteristics make poly(I:C12U) a suitable agent for use as an adjuvant in cancer directed immunotherapeutic regimes.

The rationale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes in advanced ovarian cancer.

A clinical trial employing an immunotherapeutic approach based on the use of a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes carrying tumour-associated antigens is planned in advanced ovarian cancer in conjunction with conventional first line chemotherapy. Most patients with ovarian cancer present with advanced disease and despite high initial response rate to chemotherapy the majority will relapse within 2 years with poor overall survival. Tumour antigen-specific T cells are naturally occurring in ovarian cancer patients and T cell infiltration of the tumour is highly prognostic. Novel immunotherapy to expand and activate tumour antigen-specific T cells combined with adjuvant treatment to overcome tumour-induced immunosuppression is considered to be therapeutically beneficial. The rationale for adopting such a combined approach is discussed here.

Preparation of human ovarian cancer ascites-derived exosomes for a clinical trial.

Despite initial response to chemotherapy, at least 50% of ovarian cancer patients will relapse within 18 months. Progression-free survival is related to tumour infiltration with cytotoxic T lymphocytes (CTL). We recently demonstrated that CD8+ T cell responses to recall antigens improve following tumour response to chemotherapy. Vaccination designed to expand CTL, specific for tumour-associated antigens, may be a means of improving outcome. We are planning a clinical trial in advanced ovarian cancer patients undergoing chemotherapy using a combination of a Toll-like receptor 3 (TLR3) agonist and tumour-associated ascites-derived exosomes. Tumour-derived exosomes are a potential source of tumour antigens able to induce CD8+ T cell responses when loaded on mature dendritic cells (DC). DC maturation can be achieved with Toll-like receptor (TLR) agonists, such as the GMP-grade synthetic double stranded RNA, poly[I]:poly[C12U] (Ampligen) which is a TLR-3 agonist. Here, we describe the development of a method suitable for the preparation of GMP-grade exosomes from the ascites fluid of ovarian cancer patients, and the methods used for the molecular and immunological characterisation of these exosomes preceding their use in a clinical trial.

Role of calcium chelation in high-temperature antigen retrieval at different pH values.

Recent work by Shi et al. on the mechanism of high-temperature antigen retrieval has claimed that the antigen retrieval process is pH-dependent, with different antigens benefitting at high or low pH values of antigen retrieval solutions. It has previously been claimed that chelation of Ca2+ at high temperature is an essential feature of the antigen retrieval process. In order to resolve this apparent dichotomy, the relative antigen retrieval effects were analysed using the buffers employed by Shi et al. in both a facilitating and an inhibitory mode. The results show that calcium-related effects are optimal at high pH values and do not operate at very low pH. The relative antigen retrieval effectiveness of hydrochloric acid and its metal halide solutions were also investigated in relation to pH. The results of these experiments showed that whilst HCl alone produced antigen retrieval (AR), it also produced severe tissue damage, which was reduced by the inclusion of inorganic salts. These results suggest that antigen retrieval at low pH may be achieved through the dissociation of Ca2+ complexes by high concentrations of H+ ions and/or the breaking up of cross-links from formalin fixation. Results are also presented to show that chaotropic denaturants such as urea and guanidine hydrochloride also function principally through calcium chelation, whilst detergents have no role to play in high-temperature retrieval.

Simian virus 40 large T antigen (SV40LTAg) primer specific DNA amplification in human pleural mesothelioma tissue.

BACKGROUND: DNA sequences and immunoreactivity associated with Simian virus 40 transforming factors, large T and small t antigens (SV40LTAg), suggestive of an aetiopathogenetic link have been identified in fresh frozen tissue of a high proportion of recent cases of pleural mesotheliomas from the United States, Italy and Germany. SV40 is not normally infective in man though it can transform human cells in tissue culture. A large cohort of people in the western world was accidentally parenterally inoculated with live SV40 through contaminated polio vaccines given between 1959 and 1961, and this might be a factor in the current continuing rise in the incidence of mesothelioma in the United States, Britain and Europe. The present study investigated the presence of SV40LTAg DNA in recently diagnosed cases of mesothelioma in Britain and the feasibility of detecting the SV40 DNA in archival tissue for retrospective analysis of cases in the peri-vaccination period. METHODS: DNA was extracted from fresh frozen and/or rehydrated formalin fixed, paraffin embedded tissue sections from nine recently diagnosed cases of mesothelioma, nine cases of pulmonary adenocarcinoma, and three reactive pleurae, and amplified by the polymerase chain reaction (PCR) using the primer pairs used previously on fresh frozen tissues-namely, the SV primer set directed at the LTAg gene sequence unique to SV40 and the PYV primer set directed at a sequence shared by SV40 and papovavirus strains BK and JC, respectively. RESULTS: PCR positivity with the SV primer set was restricted to four of the nine cases of mesothelioma. In contrast, six of the nine mesotheliomas, two of the nine adenocarcinomas, and one of the three reactive pleurae showed positivity with the PYV primers. The fresh frozen and corresponding formalin fixed, paraffin embedded tissue results concorded well with each other. CONCLUSIONS: Our data provide evidence for the association of SV40LTAg primer specific DNA with human pulmonary mesothelioma in the British population.

Clonal overexpression of metallothionein is induced by somatic mutation in morphologically normal colonic mucosa.

Metallothionein (MT) overexpression occurs frequently in human tumours but the underlying mechanism is unknown. Morphologically normal-appearing mucosa from human colorectal carcinoma resection specimens and of the colons of ageing laboratory mice contains scattered single crypts whose cells show uniformly increased MT immunostaining, suggesting that MT overexpression arises directly from random crypt stem cell somatic mutation, followed by colonization of the clonal unit by the mutated progeny. This hypothesis has now been tested by quantifying the frequency of immunocytochemically detectable monocryptal colorectal MT overexpression, 5 and 30 days after injection of 8-week-old mice with a single dose of the mutagen dimethylhydrazine (DMH, 30 mg/kg subcutaneous). Otherwise normal-appearing MT-positive crypts were recorded as either wholly or partially involved by the overexpressing phenotype. Five days after DMH injection, the median frequency of partially involved MT-positive crypts was 11.7 x 10(-4), declining significantly to 1.8 x 10(-4) at 30 days (Mann-Whitney U, P < 0.01). In contrast, the median frequency of wholly involved crypts was 0.2 x 10(-4) at 5 days, increasing significantly (P < 0.005) to 12.9 x 10(-4) at 30 days. The frequency of MT-positive crypts and the time course of evolution of partially involved to wholly involved forms were similar to those described for mutation-induced crypt-restricted loss of glucose-6-phosphate dehydrogenase activity in mice treated with an identical DMH regimen. The findings indicate that cellular MT overexpression can occur as a direct consequence of somatic mutation, either cis-activating mutation(s) of the MT gene itself, or trans-activating mutation(s) of other genes involved in controlling MT expression. This is likely to be an important mechanism underlying MT overexpression in neoplasia. Such mutation-induced aberrant MT expression may be involved in the acquisition of selective cellular growth of survival advantage during tumour progression.

SV40 DNA sequences in mesotheliomas.

To investigate the presence of SV40 DNA sequences in human British mesotheliomas, PCR analysis using PYV and SV primers which amplify a 172 bp fragment of SV40LTAg and a 105 bp fragment unique to the SV40LTAg respectively was performed on archival and frozen tissues. Nine pleural mesotheliomas, nine adenocarcinomas metastatic to the pleura and three inflammatory disorders of the pleura were studied. PCR positivity with the SV primer set was restricted to four of the nine cases of mesothelioma with concordance between paraffin embedded and frozen tissues. Positivity with the PYV primer set was observed in six mesotheliomas, two adenocarcinomas and one of the reactive pleurae. This study indicates that SV40 DNA sequences are present in a substantial proportion of British mesotheliomas.

Wet autoclave pretreatment for antigen retrieval in diagnostic immunohistochemistry.

Interest has recently been shown in adapting the microwave oven heating technique for antigen retrieval to routine diagnostic immunocytochemical practice. Although it has proved effective as a specialist method for individual antigen localization in many laboratories, it has certain drawbacks which have hampered its wider routine application. These include the need to monitor the sections during the microwave treatment to prevent damage or drying, the limited number of sections that can be accommodated in the microwave oven, and the inevitable alteration in nuclear morphology induced by the microwaves. In order to obviate these difficulties, we have modified the wet autoclave method of Shin et al. (Lab Invest 1991; 64: 693-702) as a routine technique for retrieval of a variety of cell surface, cytoplasmic, and nuclear antigens in formalin-fixed and paraffin-embedded tissue. The technique produces even enhancement of several refractory antigens in anatomically different sites and has the potential to handle reliably up to 200 sections at a time without significant damage to the section or to nuclear morphology.

Possible role of tissue-bound calcium ions in citrate-mediated high-temperature antigen retrieval.

High-temperature preheating of sections in the presence of a salt (e.g., citrate) or a protein denaturant (e.g., urea) solution has been shown recently to provide a reliable alternative to tissue proteolysis for antigen retrieval from formaldehyde-fixed, paraffin-embedded tissues. However, the underlying mechanism of action of this form of pretreatment remains highly speculative. In this study, we show that calcium chelating agents such EDTA and EGTA are more effective than citrate in the retrieval of a citrate-sensitive nuclear antigen, Ki-67. Also, sodium carbonate and another calcium precipitating agent are both able to effect antigen retrieval at high temperatures. The overall data therefore suggest that either the chelation or the precipitation of tissue-bound calcium ions, and perhaps also other divalent metal cations, is a critical step in salt-mediated antigen retrieval. As a corollary, it is suggested that tight complexing of calcium ions or other divalent metal cations with proteins during formaldehyde tissue fixation is responsible for the masking of certain antigens.

Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).

Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.

In vivo photoinduction of metallothionein in human skin by ultraviolet irradiation.

The aims of this study were to confirm and substantiate the in vivo cutaneous induction of metallothionein (MT) in human skin by UVR, which we have reported in brief previously, and to make a preliminary attempt to characterize the time course of this phenomenon. Buttock skin in 32 volunteers was irradiated with 2 MED of UVB and biopsies were taken at 24 h from matched non-irradiated and irradiated sites. In the kinetic study, skin biopsies from six volunteers were taken at 0, 2, 8, 24, and 48 h after 2 MED UVB irradiation. MT was immunolocalized in formalin-fixed, paraffin-embedded tissue with the monoclonal antibody E9 by an indirect immunoperoxidase method. Statistically significant differences between immunocytochemical scores were identified between non-irradiated (NI) and irradiated (I) skin within suprabasal keratinocytes (mean: NI = 1.2, I = 5.1; P = 0.01), superficial dermal fibroblasts (mean: NI = 2, I = 43; P < 0.001), mid-dermal fibroblasts (mean: NI = 0, I = 27; P < 0.001), and deep dermal fibroblasts (mean: NI = 0, I = 11; P < 0.001). In the kinetic study, no consistent rise in MT score with time was observed for the epidermal component. In dermal fibroblasts, however, the first statistically significant rise in immunocytochemically detectable MT was detected at 2 h and this was found to plateau beyond 8 h. These results confirm that ambient levels of UV irradiation are capable of inducing MT in human skin in vivo. Taken together with the relative rapidity of the response, this suggests a physiological photoprotective role for MT in human skin cells. The lack of a kinetic increase in epidermal MT may be due to high basal levels. Induction of MT in dermal fibroblasts may reflect the effects of a diffusible factor released from keratinocytes after UVR.

Clinical studies of human papilloma vaccines in pre-invasive and invasive cancer.

Cervical cancer is the second most common cause of cancer death in women worldwide. It is almost invariably associated with infection with human papilloma virus (HPV) particularly types 16 and 18. The ubiquitous expression of E6 and E7 oncogene products has been recognised as an attractive target for CTL-mediated immunotherapy. In-vivo expansion of an HPV oncogene product specific MHC class 1 restricted response has been demonstrated using intradermally administered live vaccinia virus HPV 16 and 18 E6/E7 gene construct (TA-HPV, Cantab Pharmaceuticals). Responses have been seen in 1/3 evaluable patients with advanced cervical cancer, and 3/12 CIN3 volunteers, and in 4/29 patients with early invasive cervical cancer (Rankin et al. Proceedings of 91st AACR Meeting, San Francisco, April 2000). In addition, the adoptive transfer of ex vivo HPV 16 or 18 positive autologous tumour lysate pulsed dendritic cells is currently being tested as an alternative means of expanding HPV specific CTL in advanced cervical cancer patients. So far an HLA-A*O201 restricted CD8 T cell response has been recorded in the single HLA-A*O201 patient whose tumour was shown to be HPV16 positive. It appears therefore feasible to induce HPV specific CTL responses in patients with cervical cancer using several vaccine strategies. However, further clinical trials are needed to determine the full anti-tumour potential of this vaccine based immunotherapy.