RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Antagonistas dos Receptores CCR5/farmacologia , Neoplasias Colorretais/imunologia , Cicloexanos/farmacologia , Linfócitos T Reguladores/imunologia , Triazóis/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CCL4/metabolismo , Quimiotaxia de Leucócito , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Maraviroc , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/imunologia , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Receptores CCR5/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
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.
Assuntos
Soros Imunes , Proteínas de Neoplasias/análise , Proteínas Nucleares/análise , Sequência de Aminoácidos , Animais , Biomarcadores/análise , Western Blotting , Divisão Celular , Humanos , Técnicas Imunoenzimáticas , Antígeno Ki-67 , Dados de Sequência Molecular , Neoplasias/química , Tonsila Palatina/química , OvinosRESUMO
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.
Assuntos
Adenocarcinoma/imunologia , Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Proteínas Nucleares/análise , Neoplasias Gástricas/imunologia , Adenocarcinoma/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Antígeno Nuclear de Célula em Proliferação , Neoplasias Gástricas/mortalidadeRESUMO
In a study conducted by office-based cardiologists at eight sites, 115 patients using transdermal nitroglycerin for treatment of angina pectoris participated in a four-week comparison of two transdermal nitroglycerin patches: Transderm-Nitro (TDN) and Nitro-Dur II (ND II). Patients wore two patches, one of each brand, each with half their normal prescribed daily dose of 10 mg (release rate per 24 hours) to compare patch adherence properties, patient preference, and local side effects. More ND II than TDN patches fell off (5% versus 0.1%), and fewer TDN than ND II patches became loose (1.7% versus 8.4%); 98.2% of the TDN and 86.7% of the ND II patches adhered totally. Significantly more patients expressed a preference for TDN over ND II in four of eight assessment variables, including the choice of their next prescription. The frequency of problems at the site of patch application was similar with both patches.
Assuntos
Angina Pectoris/tratamento farmacológico , Nitroglicerina/administração & dosagem , Adesividade , Administração Cutânea/efeitos adversos , Administração Cutânea/instrumentação , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Prenatal zinc deficiency and prenatal undernutrition were found to have adverse effects on the food consumption and weight gain of pregnant dams and their offspring. Pups whose dams suffered prenatal zinc deficiency (ZD) consumed less food and gained less weight than pups whose dams suffered prenatal undernutrition (PF). The PF pups consumed less food and gained less weight than pups whose dams were normally fed (AL). The ZD females at age 75 days were significantly more aggressive than the PF females, while the PF females were more aggressive than the AL females. At age 105 days, ZD females were significantly more aggressive than the PF and AL females. There were no differences in aggression between the PF and AL females at 105 days. Among the ZD, PF, and AL male offspring, there were no differences in aggression at either age level except that the 75 day old PF males were significantly less aggressive than the AL males. Thus prenatal malnutrition, especially zinc deficiency, seems to have differential effects on the aggressive tendencies of female and male offspring.
Assuntos
Agressão , Troca Materno-Fetal , Distúrbios Nutricionais/psicologia , Complicações na Gravidez , Zinco/deficiência , Fatores Etários , Animais , Peso ao Nascer , Peso Corporal , Eletrochoque , Comportamento Alimentar , Feminino , Humanos , Masculino , Gravidez , Complicações na Gravidez/psicologia , Ratos , Fatores SexuaisRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Proteínas de Ligação a DNA/análise , Proteína Potenciadora do Homólogo 2 de Zeste , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Interferon gama/biossíntese , Subunidade alfa de Receptor de Interleucina-2/análise , Leucócitos Mononucleares/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/análise , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , Proteínas Proto-Oncogênicas/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/análiseRESUMO
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.
Assuntos
Antígenos de Neoplasias/análise , Hepatite/imunologia , Neoplasias Hepáticas/secundário , Fígado/imunologia , Proteínas Nucleares/análise , Anticorpos Monoclonais/imunologia , Hepatite/patologia , Humanos , Técnicas Imunoenzimáticas , Antígeno Ki-67 , Hepatopatias/imunologia , Hepatopatias/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/análise , Antígeno Nuclear de Célula em ProliferaçãoRESUMO
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.
Assuntos
Antígenos/análise , Imuno-Histoquímica/métodos , Micro-Ondas , Neoplasias/imunologia , Feminino , Genes p53 , Humanos , Masculino , Coloração e RotulagemRESUMO
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.
RESUMO
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.
Assuntos
Anticorpos Monoclonais , Túbulos Renais/patologia , Síndrome Nefrótica/patologia , Biópsia , Divisão Celular , Humanos , Imuno-HistoquímicaRESUMO
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.
RESUMO
A total of 1525 schoolgirls aged 13 years from 21 schools in the County Borough of Dudley, were bled for titration of rubella haemagglutinating inhibiting antibody and then were immediately vaccinated with either Wistar RA 27/3 or Cendehill strain live attenuated. Both vaccines were administered subcutaneously by syringe and needle but the Wistar RA 27/3 vaccine was also given by multiple injection apparatus. Significnatly higher conversion rates and geometric mean haemagglutinating inhibiting antibody titres were obtained in girls initially seronegative given the Wister RA 27/3 than in those given the Cendehill vaccine, regardless of the method of vaccination. The RA 27/3 strain was associated with a small but significantly greater incidence of local pain immediately on injection. With this exception, differences in the occurrence of reactions were not found between vaccines, between those initially susceptible and immune or with the level of antibody response.
Assuntos
Anticorpos Antivirais/análise , Vacina contra Rubéola , Adolescente , Formação de Anticorpos , Ensaios Clínicos como Assunto , Inglaterra , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Vacina contra Rubéola/administração & dosagem , Vacina contra Rubéola/efeitos adversos , Vírus da Rubéola/imunologia , Vacinação/métodos , Vacinas AtenuadasRESUMO
A female African green monkey developed a generalized lymphosarcoma spontaneously that clinically and pathologically resembled malignant lymphoma in human beings infected with human T-lymphotropic virus type I. The monkey was serologically positive for simian T-lymphotropic virus. Immunocytochemical analysis of routinely fixed and paraffin embedded tumor tissue demonstrated neoplastic cells that stained positive with antihuman monoclonal UCHL1 which recognized a T-cell-restricted isotype of leukocyte common antigen.
Assuntos
Linfoma não Hodgkin/veterinária , Doenças dos Macacos/patologia , Animais , Chlorocebus aethiops , Feminino , Linfonodos/patologia , Linfócitos/patologia , Linfoma não Hodgkin/patologiaRESUMO
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.
Assuntos
Carcinoma/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Proteínas de Neoplasias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas , Adulto , Carcinoma/imunologia , Carcinoma/virologia , China , Regulação para Baixo , Feminino , Humanos , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/imunologia , Proteínas da Matriz Viral/metabolismoRESUMO
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.
Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/uso terapêutico , Humanos , Hibridomas/imunologia , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMO
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.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/virologia , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 4/imunologia , Leucoplasia Pilosa/imunologia , Leucoplasia Pilosa/virologia , Infecções Oportunistas Relacionadas com a AIDS/imunologia , Animais , Anticorpos Monoclonais , Células Epiteliais/imunologia , Células Epiteliais/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica , Infecções por Herpesviridae/virologia , Herpesvirus Humano 4/genética , Humanos , Mucosa Bucal/imunologia , Mucosa Bucal/patologia , Mucosa Bucal/virologia , Ratos , Latência Viral , Replicação Viral/fisiologiaRESUMO
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.
Assuntos
Apoptose/fisiologia , Proteínas de Transporte/genética , Doença de Hodgkin/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Receptor fas/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Proteínas de Transporte/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo , Doença de Hodgkin/metabolismo , Humanos , Células de Reed-Sternberg/metabolismo , Células de Reed-Sternberg/patologiaRESUMO
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.
Assuntos
Adenoviridae/genética , Neoplasias Colorretais/terapia , Escherichia coli/enzimologia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Nitrorredutases/genética , Proteínas E2 de Adenovirus/genética , Animais , Aziridinas/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Ativação Enzimática , Humanos , Camundongos , Camundongos Nus , Pró-Fármacos/metabolismo , Replicação ViralRESUMO
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.