Biomedical subjects

Peter L Stern

Publications and source records attributed to Peter L Stern.

36 records · Page 2Linked to original sources

PubMed2003

Association between human leukocyte antigen polymorphism and human papillomavirus 16-positive vulval intraepithelial neoplasia in British women.

Polymorphisms in human leukocyte antigen (HLA) genes have been implicated in the risk for developing human papillomavirus (HPV)-associated cervical neoplasia. By comparison with local cadaver controls typed for HLA class I (n = 946) and II (n = 144) antigens, HPV-16-positive high grade vulval intraepithelial neoplasia patients (n = 42) showed significantly different frequencies of HLA-A2 [odds ratio (OR), 2.1; confidence interval (CI), 1.4-3.9], HLA-B7 (OR, 2.6; CI, 1.4-4.7), HLA-DRB1*01(01/02/04) (OR, 0.1; CI, 0.03-0.5), HLA-DRB1*11 (OR, 3.3; CI, 1.4-7.1), HLA-DRB1*13 (OR, 0), HLA-DQB1*05 (OR, 0.2; CI, 0.05-0.6), and HLA-DQB1*03032 (OR, 4.6; CI, 1.5-14.0). With the exception of HLA-B7 and HLA-DRB1*11, these significant differences were also seen comparative to local HPV-16-positive cervical carcinoma patients (n = 114), suggesting a specific immunogenetic contribution that is independent of HPV-16 infection in high-grade vulval intraepithelial neoplasia. Such factors are important to the development of HPV vaccines for treatment of cervical and vulval neoplasia.

Biopsy↗

PubMed2003

No alteration in NK function or zeta chain expression in NK and T cells of cervical cancer patients.

OBJECTIVE: To investigate in vitro natural killer (NK) cell activity and expression of signal-transducing zeta chains in patients with cervical cancer. PATIENTS AND METHODS: Experiments were performed with frozen lymphocytes from patients at all disease stages and from healthy controls. Thawed NK were activated by overnight incubation in interferon-gamma (IFN-gamma); activity against two target cell lines was assessed by 4-h (51)Cr release assay. Targets chosen were K562, an erythroleukemic cell line, and a cervical carcinoma cell line designated 808. T and NK cell zeta chain expression was measured by flow cytometry. RESULTS: Patients' NK were found to be as cytotoxic as those of normal controls against cell lines K562 and 808. Patient T and NK cells did not show significant down-regulation of the zeta chain. CONCLUSIONS: We have found no evidence to suggest that loss of zeta chains is a mechanism for immunocompromise in patients with cervical carcinoma. IFN-recoverable patient NK activity is not reduced compared to matched controls. This may be clinically relevant since NK are active against cells exhibiting class I human leukocyte antigen (HLA) down-regulation and many cervical cancers show loss of HLA.

Age Factors↗

PubMed2003

Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia.

Human papillomavirus type 16 (HPV-16)-associated vulval intraepithelial neoplasia (VIN) is frequently a chronic, multifocal high-grade condition with an appreciable risk of progression to vulval cancer. The requirement to treat women with VIN has recently stimulated the use of immunotherapy with E6/E7 oncogene vaccines. Animal models have shown that E2 may also be a useful vaccine target for HPV-associated disease; however, little is known about E2 immunity in humans. This study investigated the prevalence of HPV-16 E2-specific serological and T-cell responses in 18 women with HPV-16-associated VIN and 17 healthy volunteers. E2 responses were determined by full-length E2-GST ELISA with ELISPOT and proliferation assays using E2 C-terminal protein. As positive controls, HPV-16 L1 responses were measured using virus-like particles (VLPs) and L1-GST ELISA with ELISPOT and proliferation using VLPs as antigen. The VIN patients all showed a strong serological response to L1 compared with the healthy volunteers by VLP (15/18 vs 1/17, P<0.001) and L1-GST ELISA (18/18 vs 1/17, P<0.001). In contrast, L1-specific cellular immune responses were detected in a significant proportion of controls but were more prevalent in the VIN patients by proliferation assay (9/17 vs 17/18, P<0.02) and interferon-gamma ELISPOT (9/17 vs 13/18, P=not significant). Similar and low numbers of patients and controls were seropositive for E2-specific Ig (2/18 vs 1/17). In spite of previous studies showing the immunogenicity of E2 in eliciting primary T-cell responses in vitro, there was a low prevalence of E2 responses in the VIN patients and controls (2/18 vs 0/17).

Adult↗

PubMed2002

Characterization of the murine 5T4 oncofoetal antigen: a target for immunotherapy in cancer.

Human 5T4 oncofoetal antigen defined by the murine 5T4 monoclonal antibody is a highly glycosylated protein expressed by trophoblast and a few specialized adult epithelia. Up-regulation of 5T4 expression in some cancers is associated with poor clinical outcome; overexpression of human 5T4 cDNA in epithelial cells can alter their morphology and motility, supporting a role for such functions in cancer and development. A murine model to study 5T4 biology and tumour immunology would be useful. The production of m5T4-specific antibodies, their use in establishing transfected cells and documenting their biological properties in vitro are described. A rat monoclonal antibody specific for mouse 5T4 molecules by ELISA, flow cytometry, immunohistochemistry and immunoprecipitation was isolated and epitope mapped. Similar to its human counterpart, murine 5T4 antigen is a 72 kDa glycoprotein (immunoprecipitation and Western blot analysis) and exhibits punctate cell surface expression, dependent upon the integrity of the actin cytoskeleton. Likewise, overexpression of autologous murine 5T4 by B16 F10 melanoma cells and A9 L fibroblasts accentuates the 5T4 phenotype, which is characterized by a spindle-like morphology, increased motility, and reduced adhesion and proliferation rate. Immunohistochemical analysis of adult mouse tissues shows a restricted pattern of expression similar to that of human 5T4 antigen. The murine 5T4 antigen-expressing cell lines and antibody reagents are now being used to explore novel immunotherapies in pre-clinical models and the biology of 5T4 in development.

Animals↗

PubMed2002

Immunization with a recombinant adenovirus encoding a lymphoma idiotype: induction of tumor-protective immunity and identification of an idiotype-specific T cell epitope.

The Ig Id of a B cell lymphoma is a tumor-specific Ag, although as a self-Ag it is likely to be a weak immunogen. Provision of a foreign gene may enhance the immunogenicity of the idiotype. Viral vectors allow highly efficient transfer of genetic material and are themselves innately immunogenic. We have investigated the ability of recombinant adenoviral vectors, encoding the idiotypic gene with or without fusion to the human Fc region, to produce anti-idiotypic Ab- and T cell-mediated responses in a syngeneic BALB/c A20 murine lymphoma model. The idiotypic V(H) and V(L) sequences were assembled as a single chain variable fragment (scFv) and adenoviral vectors encoding the A20 scFv (Ad.A20) and A20 scFv linked to the Fc fragment of human IgG1 (Ad.A20hFc) were constructed. A single immunization of BALB/c mice with Ad.A20hFc but not Ad.A20 induced a specific anti-idiotypic Ab response. T cell lines generated from mice vaccinated with either vector displayed specific cytotoxicity, proliferation, and IFN-gamma release against a syngeneic dendritic cell line transduced using a retroviral vector to express the A20 scFv idiotype (XS52.A1.A20). Importantly, both T cell lines lysed the A20 lymphoma cells. An immunodominant H-2K(d)-restricted CD8(+) T cell peptide, DYWGQGTEL (A20[106-114]), was identified as a naturally occurring A20 scFv epitope. A single immunization with Ad.A20hFc but not Ad.A20 provided protection in >40% of animals challenged with a lethal dose of the A20 tumor line and was more effective, in this model, than a previously optimized plasmid vaccine.

Adenoviridae↗

PubMed2002

Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen.

The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.

Animals↗

PubMed2002

5T4 interacts with TIP-2/GIPC, a PDZ protein, with implications for metastasis.

Overexpression of the 5T4 transmembrane glycoprotein can have marked effects on both the actin cytoskeleton and cell migration. Using a yeast two-hybrid approach, we describe a novel interaction between 5T4 and TIP-2/GIPC, a cytoplasmic interacting protein containing a PDZ domain. The cytoplasmic tail of 5T4 contains a class I PDZ-binding motif (Ser-Asp-Val) and we demonstrate that this region, in particular the terminal valine, is required for 5T4 interaction with TIP-2/GIPC. HeLa cells expressing hemagglutinin-tagged TIP-2/GIPC (HA-TIP-2/GIPC) have an altered distribution of endogenous 5T4, which colocalizes with HA-TIP-2/GIPC, thus supporting an interaction. Furthermore, TIP-2/GIPC can be coimmunoprecipitated with 5T4 from HeLa cell lysates. Identification of the 5T4 and TIP-2/GIPC interaction provides the first link between 5T4 and the actin cytoskeleton. Since other proteins, like 5T4, associate with TIP-2/GIPC and are linked with cancer, we explore the possibility that TIP-2/GIPC may be a common factor involved in the cancer process.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Targeting immune effector molecules to human tumor cells through genetic delivery of 5T4-specific scFv fusion proteins.

Although several clinical trials have shown beneficial effects by targeting tumor-associated antigens (TAAs) with monoclonal antibodies, a number of issues, including poor penetration of the tumor mass and human antimouse antibody responses, remain. The use of recombinant single-chain Fv (scFv) fragments has the potential to address these and other issues while allowing the addition of different effector functions. To develop therapeutic strategies that recruit both humoral and cellular arms of the immune response, we have constructed chimeric proteins linking either the human IgG1 Fc domain or the extracellular domain of murine B7.1 to a scFv specific for the oncofetal glycoprotein, 5T4. This TAA is expressed by a wide variety of carcinomas and is associated with metastasis and poorer clinical outcome. We have engineered retroviral constructs that produce fusion proteins able to interact simultaneously with both 5T4-positive cells and with the receptor/ligands of the immune effector moieties. Genetic delivery through a murine leukemia virus vector to 5T4-positive tumor cells results in the secreted scFv fusion protein binding to the cell surface. Furthermore, the scFv-HIgG1 fusion protein is able to direct lysis of 5T4-expressing human tumor cell lines through antibody-dependent cell cytotoxicity, indicating its potential as a gene therapy for human cancers.

Antibody-Dependent Cell Cytotoxicity↗

PubMed2002

The use of vaccines in the prevention and treatment of cervical cancer.

The close association between high risk HPV infection and cervical carcinoma has provided the impetus for the development of prophylactic and therapeutic vaccination schedules. An effective prophylactic vaccine would obviate the need for population-based cervical screening programmes, while therapeutic vaccination might provide an effective adjunct to or replacement for conventional treatment for benign and malignant cervical disease. While the challenges associated with the design and implemention of immunotherapies are numerous, optimism remains high and it is expected that the next few decades will witness a revolutionary change in the way we treat cervical cancer and its premalignant lesions. A papillomavirus vaccine that prevented HPV infection on the one hand and acted against established disease on the other, would have a profound impact on one of the major cancers affecting women globally.

Animals↗

PubMed2002

Cancer vaccines and immunotherapy.

It is now clear that many human tumour antigens can be recognised by the immune system. These tumour antigens can be classified into several groups including cancer-testis, differentiation, tissue specific, over-expressed, and viral-associated antigens. In many cases, there is a known molecular basis of carcinogenesis which provides the explanation for the differentiated expression of these antigens in tumours compared with normal cells. Improved understanding of the biology of the immune response, particularly of immune recognition and activation of T-cells, allow better design of vaccines. Pre-clinical comparative studies allow evaluation of optimal vaccine strategies which can then be delivered to the clinic. Currently, a range of cancer vaccines are being tested including those using tumour cells, proteins, peptides, viral vectors, DNA or dendritic cells. Ultimately, this research should give rise to an entirely new modality of cancer treatments.

Antigens, Neoplasm↗

PubMed2002

The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells.

It has been reported recently that the cytomegalovirus (CMV) immediate-early promoter is transcriptionally inactive in undifferentiated mouse embryonic stem (ES) cells. This result is surprising, since the CMV promoter is used to express transgenes in a variety of cell lines. We studied the expression of a human CMV-driven enhanced green fluorescent protein (EGFP) reporter gene (pEGFP-N1) in five undifferentiated mouse ES cell lines (BL/6III, D3, E14TG2a, MESC20, and 129) and found EGFP expression in all of these cell lines. Under optimal conditions, between 50%-80% transfection efficiencies could be achieved, and EGFP expression levels were maintained for at least 72 hours. Therefore, the human CMV promoter remains a useful system for transgene expression in undifferentiated ES cells.

Animals↗

PubMed2002

Safety and immunogenicity of TA-HPV, a recombinant vaccinia virus expressing modified human papillomavirus (HPV)-16 and HPV-18 E6 and E7 genes, in women with progressive cervical cancer.

PURPOSE: Cervical cancer, the second most common malignancy in women worldwide, is almost invariably associated with infection by human papillomavirus (HPV). HPV-16 or -18 is commonly present in 70% of cervical cancers. HPV-positive tumor cells present antigens of the viral protein in the context of human leukocyte antigen (HLA) class I that can be recognized by CTLs. We have conducted a study in patients with early-stage cervical cancer to assess the safety and immunological effects of vaccination with TA-HPV, a live recombinant vaccinia virus expressing modified forms of the HPV-16 and -18 E6 and E7 proteins. EXPERIMENTAL DESIGN: Twenty-nine patients with clinical International Federation of Gynecologists and Obstetricians (FIGO) stage Ib or IIa cervical cancer were given two vaccinations with TA-HPV at least 4 weeks apart, starting 2 weeks before radical hysterectomy. Patients were monitored closely for side effects of the vaccination. Serial blood samples were examined for HPV-specific CTLs or changes in levels of antibodies to HPV-16 or -18 E6 and E7 proteins and to vaccinia virus. RESULTS: Vaccination with recombinant vaccinia was well tolerated in all patients with only mild to moderate local toxicity, and no serious adverse events were attributable to the vaccine. After a single vaccination, HPV-specific CTLs were found in four patients (HLA A1, A3, three patients; HLA A1, A24, one patient). Eight patients developed HPV-specific serological responses. CONCLUSIONS: This study confirmed the safety and immunogenicity of the vaccine in a proportion of those patients vaccinated. Additional clinical studies using TA-HPV in combination with an additional experimental vaccine for HPV-16 are currently under way.

Adenocarcinoma↗

PubMed2002

Attenuated recombinant vaccinia virus expressing oncofetal antigen (tumor-associated antigen) 5T4 induces active therapy of established tumors.

The human oncofetal antigen 5T4 (h5T4) is a transmembrane glycoprotein overexpressed by a wide spectrum of cancers, including colorectal, ovarian, and gastric, but with a limited normal tissue expression. Such properties make 5T4 an excellent putative target for cancer immunotherapy. The murine homologue of 5T4 (m5T4) has been cloned and characterized, which allows for the evaluation of immune intervention strategies in "self-antigen" in vivo tumor models. We have constructed recombinant vaccinia viruses based on the highly attenuated and modified vaccinia virus ankara (MVA strain), expressing h5T4 (MVA-h5T4), m5T4 (MVA-m5T4), and Escherichia coli LacZ (MVA-LacZ). Immunization of BALB/c and C57BL/6 mice with MVA-h5T4 and MVA-m5T4 constructs induced antibody responses to human and mouse 5T4, respectively. C57BL/6 and BALB/c mice vaccinated with MVA-h5T4 were challenged with syngeneic tumor line transfectants, B16 melanoma, and CT26 colorectal cells that express h5T4. MVA-h5T4-vaccinated mice showed significant tumor retardation compared with mice vaccinated with MVA-LacZ or PBS. In active treatment studies, inoculation with MVA-h5T4 was able to treat established CT26-h5T4 lung tumor and to a lesser extent B16.h5T4 s.c. tumors. Additionally, when C57BL/6 mice vaccinated with MVA-m5T4 were challenged with B16 cells expressing m5T4, resulting growth of the tumors was significantly retarded compared with control animals. Furthermore, mice vaccinated with MVA-m5T4 showed no signs of autoimmune toxicity. These data support the use of MVA-5T4 for tumor immunotherapy.

Animals↗

PubMed1999

Human papillomavirus type 16 E6 variants in cervical carcinoma: relationship to host genetic factors and clinical parameters.

Infection with human papillomavirus type 16 (HPV-16) confers a high risk for the development of cervical neoplasia. Variants of this virus may interact differentially with host genetic factors, possibly altering the disease course. Thus, HPV-16 E6 variants may differ in their ability to degrade p53 whereas the polymorphic p53 alleles may provide more or less susceptible substrates for the viral oncogene product. Also, E6 variants may differ in immunogenicity by generating different peptides for presentation by polymorphic HLA molecules to specific T cells. This study examines HPV-16 E6 sequence variation in cervical carcinomas from the UK and its relationship to polymorphism of HLA and p53 and to clinical parameters. Sequence analysis of the HPV-16 E6 ORF from 77 tumour biopsies detected the viral prototype sequence in 38% of cases. The most common variation detected was a T to G transition at base pair 350, resulting in an amino acid change from a leucine to a valine. Overall, the frequencies of 350T and 350G sequences were similar (49. 4% and 50.6% respectively). Other mutations of lower frequencies were detected together with and independently of 350G. HPV-16 E6 sequence variation at base pair 350 did not correlate with HLA genotype or clinical outcome. There was no difference in the distribution of p53 proline and arginine alleles between HPV-16-positive cervical carcinoma patients and local controls, and no influence on clinical outcome; however, there was a trend for an increased frequency of p53 arginine homozygotes among the 350T carcinoma patients.

Adult↗

PubMed1999

Human papillomavirus type 16 E2-specific T-helper lymphocyte responses in patients with cervical intraepithelial neoplasia.

T-cell-mediated immune responses against mucosal oncogenic types of human papillomaviruses (HPV) are thought to play a role in the control of the virus infection and its associated cervical lesions. The in vitro production of interleukin-2 by T-helper (Th) cells in response to the C-terminal and N-terminal domains of the HPV-16 E2 protein was determined in 74 women with cytological evidence of premalignant cervical epithelial neoplasia who participated in a non-intervention follow-up (FU) study. Cross-sectional analysis at the end of FU showed that Th cell responses against the C-terminal domain were associated with evidence of previous or present HPV-16 infection as compared to patients with no evidence of any HPV infection (18.9% versus 0%, P = 0.039). Th cell responses against the N-terminal domain were not associated with evidence of HPV-16 infection. No association with disease outcome was observed with Th cell responses against either of the E2 protein domains. However, longitudinal analysis revealed that Th cell responses against the C-terminal domain frequently occur at the time of virus clearance. Whether these responses are responsible for the clearance of the virus is not known.

Adult↗

PubMedDate not supplied

Adoptive transfer of anti-idiotypic T cells cure mice of disseminated B cell lymphoma.

There is extensive interest in idiotypic vaccination as a treatment of lymphoma. An alternative approach is the adoptive transfer of in vitro generated T cells. This strategy has been used to treat posttransplantation EBV-related diseases. The ability to generate in vitro T cells to peptides derived from immunoglobulin idiotypes raises the possibility of directly using such cells as a treatment of lymphoma. Investigating the adoptive transfer of specific T cells to idiotype derived peptides in a murine lymphoma model is therefore an important part of the clinical translation of this alternative approach. We have generated an idiotype-specific T cell line, able to recognise a defined, naturally processed idiotype-derived epitope. This line has been used to successfully treat mice with disseminated lymphoma supporting the clinical use of idiotype specific T cells.

Adenoviridae↗

PubMedDate not supplied

The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens.

Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3zeta chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.

Antigens, CD19↗

PubMedDate not supplied

Oncofetal antigen 5T4 expression as a prognostic factor in patients with gastric cancer.

BACKGROUND: Oncofetal antigen 5T4 is defined by a monoclonal antibody raised against placental trophoblast membranes. Antigen 5T4 is reported to be expressed only on trophoblasts and neoplastic cells and would be a potentially useful marker for neoplastic cells. MATERIALS AND METHODS: We evaluated the expression of 5T4 oncofetal antigen in gastric cancer by immunohistochemistry and investigated whether or not the expression of this antigen is correlated with the prognosis of patients with gastric cancer. RESULTS: The 5T4 antigen was detected in 51.6% of primary gastric cancer (n = 62) and 5T4-reactivity was more frequently observed in carcinoma with lymph node metastasis (p < 0.05). The overall survival of 5T4-positive patients was significantly worse than that of 5T4-negative patients, analyzed by the log-rank test (p < 0.05), although multivariate analysis indicated that 5T4 expression was not an independent prognostic factor. CONCLUSION: The assessment of 5T4 expression may be useful in identifying gastric cancer patients with a poor prognosis.

Aged↗