The effects of CCR5 inhibition on regulatory T-cell recruitment to colorectal cancer.

BACKGROUND: Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5-/- mice, associated with reduced tumour Treg infiltration. METHODS: Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models. RESULTS: Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5(high) Treg were more suppressive than their CCR5(low) Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth. CONCLUSIONS: CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.

The polycomb group proteins, BMI-1 and EZH2, are tumour-associated antigens.

We used SEREX technology to identify novel tumour-associated antigens in patients with primary hepatocellular carcinoma and found serological responses to the polycomb group (PcG) protein BMI-1, which is overexpressed in a range of different tumour types. Further studies identified T-cell responses to both BMI-1 and another PcG protein, EZH2, in cancer patients and at relatively lower levels in some normal donors. We next identified several CD8+ T-cell epitopes derived from BMI-1 and EZH2 and demonstrated that EZH2-derived peptides elicited more significant interferon-gamma (IFN-gamma) release than BMI-1-derived peptides. That CD8(+) T cells were responsible for the observed responses was confirmed for EZH2 by both IFN-gamma capture assays and tetramer staining using an HLA-A0201-restricted, EZH2-derived YMSCSFLFNL (aa 666-674) epitope. The ability of YMSCSFLFNL (aa 666-674) to stimulate the in vitro expansion of specific T cells from peripheral blood lymphocytes was greatly enhanced when the CD25(+) T-cell population was depleted. EZH2-specific cytotoxic T lymphocyte clones specific for two HLA-A0201 epitopes were generated and found to recognise endogenously processed EZH2 in both HLA-matched fibroblasts and tumour cell lines. Given the widespread overexpression of PcG proteins in cancer and their critical role in oncogenesis, these data suggest that they may be useful targets for cancer immunotherapy.

Enhanced efficacy of Escherichia coli nitroreductase/CB1954 prodrug activation gene therapy using an E1B-55K-deleted oncolytic adenovirus vector.

Viruses that replicate selectively in cancer cells constitute an exciting new class of anticancer agent. The conditionally replicating adenovirus (CRAd) dl1520, which lacks the E1B-55K gene, has elicited significant clinical responses in humans when used in combination with chemotherapy. A convergent development has been to use replication-defective viruses to express prodrug-activating enzymes in cancer cells. This can sensitize the cancer to prodrug, but depends upon achieving sufficient level, distribution and specificity of enzyme expression within the tumour. In this study, we have expressed the prodrug-activating enzyme nitroreductase (NTR) in the context of an E1B-55K-deleted adenovirus, CRAd-NTR(PS1217H6). We show that CRAd-NTR(PS1217H6) retains oncolytic growth properties, and expresses substantially more NTR than a comparable, replication-defective adenovirus. The combination of viral oncolysis and NTR expression results in significantly greater sensitization of SW480 and WiDr colorectal cancer cells to the prodrug CB1954 in vitro. In vivo, CRAd-NTR(PS1217H6) was shown to replicate in subcutaneous SW480 tumour xenografts in immunodeficient mice, resulting in more NTR expression and greater sensitization to CB1954 than with replication-defective virus. Combination therapy of CRAd-NTR(PS1217H6) with CB1954 reduced tumour growth from 13.5- to 2.8-fold over 5 weeks, and extended median survival from 42 to 81 days, compared with no treatment.

Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.

Hodgkin's lymphoma (HL) is characterized by the presence of malignant so-called Hodgkin's/Reed-Sternberg (HRS) cells, which display resistance to certain apoptotic stimuli, including a lack of sensitivity to Fas-mediated cell death. However, the mechanisms responsible for their resistance to apoptosis inducers have not been elucidated. Here we confirm that both HL-derived cell lines and the HRS cells of primary HL tissues express Fas ligand (FasL) along with the inhibitory c-FLIP protein. Down-regulation of cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) through the use of specific small inhibitory RNAs (siRNAs) leads to reduced viability of the L428 and L591 HL-derived cell lines. To determine whether endogenous FasL was responsible for the reduction in cell viability observed after down-regulation of c-FLIP, L428 and L591 cells were treated with c-FLIP-specific siRNAs with and without siRNAs directed to FasL. Treatment of these cells with both c-FLIP- and FasL-specific siRNAs in combination restored cell viability to near control levels. Our results provide a mechanism whereby HRS cells are protected from autonomous FasL-mediated cell death while preserving their ability to evade immunosurveillance. Targeting c-FLIP could provide a novel approach to the treatment of HL.

Heterogeneity of HLA and EBER expression in Epstein-Barr virus-associated nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an aggressive tumour of multifactorial aetiology that, although rare in most parts of the world, poses a significant mortality problem in its high incidence area of Southern China. Improved therapies are an urgent requirement and, towards this end, immunotherapeutic methods are being developed in several centres. Such strategies are dependent on the immune competence of the target tumour, in particular its expression of HLA class-I. We examined HLA class-I and -II expression in 27 primary NPC biopsies and found that 15% were extensively down-regulated for class-I expression with the majority of tumour cells appearing negative. Whilst HLA class-II was expressed at high levels in the majority of tumours, 37% showed substantial down-regulation. NPC is associated with Epstein-Barr virus (EBV). Expression of the virus-encoded EBER RNAs is accepted as a marker of EBV latency and is regarded as a valuable diagnostic criterion. EBER RNAs were expressed in all samples, but in some the level was remarkably heterogeneous, being barely detectable in many tumour cells. Our study reinforces the concept of extensive phenotypic variation in NPC. There are morphological differences between tumour cells. Some tumours express HLA class-I and/or -II, whilst others are down-regulated or negative. Individual tumours may or may not express the EBV-encoded LMP-1 protein, and individual tumour cells may express high levels of EBER, yet adjacent tumour cells express very little or none.

Monoclonal antibodies.

Monoclonal antibodies are essential tools for many molecular immunology investigations. In particular, when used in combination with techniques such as epitope mapping and molecular modelling, monoclonal antibodies enable the antigenic profiling and visualisation of macromolecular surfaces. In addition, monoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents. Furthermore, the continuous culture of hybridoma cells that produce these antibodies offers the potential of an unlimited supply of reagent. In essence, when compared with the rather limited supply of polyclonal antibody reagents, the feature of a continuous supply enables the standardisation of both the reagent and the assay technique. Clearly, polyclonal and monoclonal antibodies have their advantages and disadvantages in terms of generation, cost, and overall applications. Ultimately, monoclonal antibodies are only produced when necessary because their production is time consuming and frustrating, although greatly rewarding (at least most of the time!). This is especially apparent when a monoclonal antibody can be applied successfully in a routine pathology laboratory or can aid in the clinical diagnosis and treatment of patients. In this article, the generation and application of monoclonal antibodies are demystified to enable greater understanding and hopefully formulate novel ideas for clinicians and scientists alike.

Overexpression of p53 protein in primary Ewing's sarcoma of bone: relationship to tumour stage, response and prognosis.

Biopsy tissues of 52 patients with Ewing's sarcoma of bone treated between 1983 and 1993 were examined immuno-histochemically to determine the significance of p53 protein in diagnosis and prognosis of Ewing's sarcoma. Mean age at diagnosis was 17 years (range 6-36) and minimum follow-up was 30 months. The tumours were located in the extremities and central bones in 35 and 17 patients respectively. Metastases were present in seven patients at diagnosis. Treatment consisted of chemotherapy, surgery and/or radiotherapy in all the patients. Overexpression of p53 protein was demonstrated in seven patients (14%). There was no relationship between expression of p53 and site of tumours. Patients who overexpressed p53 protein appeared to have more advanced diseases at diagnosis and poorer response to chemotherapy than those without p53 overexpression. The 5-year relapse-free survival and overall survival in patients without metastases at the time of diagnosis were 66% and 71%, respectively, in p53 protein-negative patients compared with 20% relapse-free and overall survival in those with p53 protein overexpression (P= 0.01). The poorer prognosis in p53 protein-positive patients was independent of site, local treatment or necrosis of the tumours (P < 0.05). Over-expression of p53 protein is an independent poor prognostic factor in Ewing's sarcoma of bone.

The expression of the Epstein-Barr virus nuclear antigen (EBNA-I) in oral hairy leukoplakia.

OBJECTIVE: Oral hairy leukoplakia (OHL) is characterised by the presence of a replicative Epstein-Barr virus (EBV) in the superficial layers of the epithelium. There is some doubt, however, whether this reflects activation of a latent infection of the basal epithelial cells. EBV latency is associated with the expression of the viral gene product EBNA-I and the aim of this study was to investigate EBNA-I expression in OHL. METHODS: 22 biopsies of clinically suspicious OHL and three cases of normal mucosa were available as fresh frozen or paraffin embedded material. EBNA-I was detected immunocytochemically using a rat monoclonal antibody (IH4-I) following microwave irradiation. Lytic EBV infection was confirmed by the identification of the BZLF-I protein. RESULTS: 16 of the 22 cases displayed focal replicative EBV meeting the criteria for OHL, and in 13 of these, EBNA-I expression was restricted to the nuclei of epithelial cells in the upper layers of the epithelium. EBNA-I expression was absent from the basal cells in all cases and in the nine BZLF-I negative mucosal biopsies. CONCLUSION: These findings suggest that lytic EBV infection in OHL is not the result of activation of a latent infection of basal cells and suggests a role for EBNA-I, not only in latent EBV infection, but also in virus replication.

Simple method for pretreatment of tissue sections for the detection of apoptosis by in situ end-labelling and in situ nick translation.

Aims-To overcome the problems associated with proteolytic pretreatment of tissue sections for the detection of apoptosis.Methods-Formalin fixed, paraffin wax embedded tissue sections of reactive lymph nodes and biopsy specimens of Burkitt lymphoma were pretreated by pressure cooking for the detection of apoptosis using the in situ end-labelling and in situ nick translation methods.Results-The results achieved with the in situ end-labelling and nick translations methods were compared with those obtained using a novel anti-apoptosis specific protein (ASP) antibody. The staining patterns generated using the three methods were similar and consistent, although the ASP antibody seemed to be more sensitive and detected higher numbers of apoptotic cells within sections.Conclusions-Pressure cooking is advocated as an alternative method to proteolytic enzyme digestion for pretreating paraffin wax sections. It is reliable, inexpensive, reduces the need to optimise pretreatment variables for different tissues, and permits double immunostaining of sections.

In situ detection of the Epstein-Barr virus-encoded nuclear antigen 1 in oral hairy leukoplakia and virus-associated carcinomas.

A new monoclonal antibody has been used to examine immunohistochemically the expression of the Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA) 1 in virus-associated epithelial lesions. EBNA1 was detected in the tumour cell nuclei of 10/13 undifferentiated nasopharyngeal carcinomas and of 10/10 EBV-associated gastric carcinomas. EBNA1 was also detected in 13 of 16 oral hairy leukoplakia (HL) samples, where its expression was confined to nuclei in the upper epithelial cell layers whilst basal epithelial cells were negative. This observation is in agreement with previous studies demonstrating the absence of latent EBV infection in the basal cell compartment of HL and suggests an essential role for EBNA1, not only in latent EBV infection but also in virus replication.

Detection of Ki67 antigen by a new sheep polyclonal antiserum.

This report describes the characterisation of a polyclonal sheep antiserum against the Ki67 antigen. On western blots, this antiserum recognises a pair of bands of high molecular weight identical with those seen with another polyclonal Ki67 antiserum and the MIB 1 monoclonal antibody. The new antiserum showed nuclear staining of a proportion of cells in paraffin wax embedded tissue sections following antigen retrieval using a microwave oven or pressure cooker. This staining pattern was blocked by incubating the serum with the peptide used as immunogen. The proportion and distribution of immunostained nuclei was identical with that seen with the alternative reagents that recognise the Ki67 antigen. The new reagent stained the same proportion of cells when used over a wide range of dilutions. There was no cross-reactivity with unrelated antigens sometimes detected by the monoclonal antibodies.

Measurement of proliferation in renal biopsy specimens: evidence of subclinical tubular damage in the nephrotic syndrome.

BACKGROUND: Proliferating cells can be detected in histological material in various ways. This investigation was to study the feasibility and usefulness of application of proliferation markers to routine renal biopsy specimens. METHODS: One hundred and thirteen renal biopsies fixed in formalin and embedded in paraffin were studied immunohistologically using antibody MIB 1, which recognises the Ki-67 antigen. Twenty-two biopsies were also immunostained with antibody PC10 against proliferating cell nuclear antigen. RESULTS: PC10 stained nuclei in all biopsies, including those that were negative with MIB 1. MIB 1 stained nuclei in endocapillary sites to various extents, and there was an average of less than one stained endocapillary nucleus per glomerulus in biopsies with IgA nephropathy and IgM nephropathy, conventionally regarded as types of proliferative glomerulonephritis. Glomerular extracapillary nuclei were stained by MIB 1 in vasculitic disorders and at the site of tip changes. MIB 1 also stained nuclei in the arterial intima, especially in vascular rejection, and in interstitial tissues, correlating with renal excretory function. Tubular nuclei stained by MIB 1 were common in biopsies from patients with renal impairment and in a group with the nephrotic syndrome, in many of whom renal function was normal. CONCLUSIONS: The main conclusions are that (1) immunohistological markers of proliferation can be applied to routine renal biopsy material; (2) PC10 appears to overestimate proliferation compared with MIB 1; and (3) there is evidence of subclinical tubular damage in the nephrotic syndrome, shown by increased tubular proliferation without clinical renal impairment. This observation seems not to have been made previously.

Expression of B7 (CD80) and CD40 antigens and the CD40 ligand in Hodgkin's disease is independent of latent Epstein-Barr virus infection.

Aim-To examine the expression of CD40 and B7 (CD80) antigens and the CD40 ligand in Hodgkin's disease.Methods-Antigen and ligand expression was studied in 17 cases of Hodgkin's disease using immunohistochemistry. The study included 11 cases of Hodgkin's disease in which latent Epstein-Barr virus (EBV) infection could be demonstrated within tumour cells by in situ hybridisation for the EBV encoded early RNAs (EBERs).Results-In all cases, irrespective of EBV status, Reed-Sternberg cells and their variants (HRS cells) showed strong expression of both B7 and CD40 antigens. CD40 ligand expression was not shown in HRS cells but was confined to a subset of small lymphocytes some of which were seen to be in intimate contact with HRS cells.Paraffin wax sections from a further 60 cases of Hodgkin's disease were examined for CD40 and EBER expression alone. The CD40 antigen was identified in HRS cells in all of these cases irrespective of EBER expression.Conclusions-As CD40 and B7 expression are features of professional antigen presenting cells, these results provide further evidence that HRS cells may have antigen presenting properties and that this may contribute to the characteristic recruitment and activation of non-malignant lymphocytes which is a feature of Hodgkin's disease. The ability of HRS cells to activate T(h) cells may in turn contribute to their own survival through the induction of the gp39/CD40 pathway.

Stathmin expression is a feature of proliferating cells of most, if not all, cell lineages.

BACKGROUND: Stathmin is a phylogenetically conserved protein which was identified initially as a prominent cytosolic protein in hemopoietic cells, endocrine cells, brain, and testis. In these tissues, it has been suggested that the level of stathmin expression is important in development and cell proliferation. Furthermore, stathmin phosphorylation appears to be involved in the regulation of cell growth arrest, terminal differentiation, and hormone secretion. Elevated levels of expression of stathmin have been described in leukemia and lymphoma cells. The aim of this study was to characterize the distribution of cells that express the stathmin protein in a wide variety of normal human and rodent tissues. EXPERIMENTAL DESIGN: First, antisera against a synthetic stathmin peptide have been raised in rabbits and the specificity of these antisera confirmed by their reactivity with stathmin on 1-dimensional and 2-dimensional Western blots. Second, the most appropriate means of fixing tissues in order to stain stathmin has been investigated. Finally, using the optimized conditions of tissue fixation, the antisera have been used to immunostain sections taken from a wide variety of tissues. RESULTS: Immunopositivity was found in cells of all the lineages studied, with the stained cells present within the proliferating compartment of tissues. Conversely, most nonproliferating mature cells did not stain with the antisera to stathmin. The only nonproliferating cells that appeared to express stathmin were a subpopulation of glial cells, neurons, and anterior pituitary cells. CONCLUSIONS: It is proposed that stathmin is necessary for cell proliferation in most, or all, cell lineages and that its primary function relates to some aspect of cell division.

Microwave oven antigen retrieval applied to the immunostaining of cytopathology specimens.

Microwave oven antigen retrieval has been developed to extend the range of antibodies that can be used upon sections of fixed and processed tissue. It has the additional advantages of improving immunostain intensity and reducing background positivity. It can also be employed as an alternative to proteolytic digestion. In this study the effects of microwave oven heating upon immunochemical staining of cytopathological specimens with a range of selected antibodies have been investigated. Microwaving did not result in loss of cells, and there was no need to use adhesive-coated slides. The method improved the staining intensity and reduced background with antibodies against a variety of antigens that are difficult or impossible to detect in formaldehyde-fixed cytological material. Microwave heating was also used successfully as an alternative to trypsin digestion, and had the advantage of reduced morphological distortion. The technique was useful in demonstrating the soluble formalin-sensitive antigen p19 on cytospins fixed in formaldehyde vapour. Microwave oven heating thus shows promise of extending the scope of immunostaining in clinical cytopathology.

Expression of proliferating cell nuclear antigen (PCNA) in gastric carcinoma: no evidence for prognostic value.

It has been proposed that immunostaining with PC10, a monoclonal antibody against proliferating cell nuclear antigen (PCNA), is of prognostic value in gastric carcinoma. Gastric carcinomas from a series of 90 patients in whom survival data were known have been studied. There was no relation between the degree of PC10 immunostaining assessed semiquantitatively and survival.

Immunohistochemical evidence for the expression of proliferating cell nuclear antigen (PCNA) by non-proliferating hepatocytes adjacent to metastatic tumours and in inflammatory conditions.

The possibility that proliferating cell nuclear antigen (PCNA) is expressed by non-proliferating liver cells was investigated. Liver biopsies from 107 patients were investigated, which included histologically normal liver, metastatic tumour, and inflammatory lesions. PCNA was detected using immunohistochemical staining with the monoclonal antibody PC10. This was compared with the proportion of proliferating cells as assessed by immunostaining for the Ki-67 antigen using the monoclonal antibody MIB 1. Most cases of histologically normal liver showed few PC10-positive cells. PCNA-positive hepatocytes far outnumbered those positive with MIB 1 in specimens showing metastatic tumour or an inflammatory cell infiltrate. There was no relation between the degree of PCNA overexpression and the type of tumour present or the nature of the inflammatory lesion. Other cell types, including the biliary epithelium, did not show this large difference between the proportions of PC10- and MIB 1-positive cells. It is concluded that non-proliferating hepatocytes increase their levels of PCNA in a wide variety of pathological conditions. This may be mediated by cytokines released by tumour cells or inflammatory cells.