Role of alkaline phosphatase in colitis in man and rats.

BACKGROUND AND AIMS: Crohn's disease (CD) and ulcerative colitis (UC) are chronic multifactorial inflammatory bowel diseases (IBDs) with unknown aetiology, but a deregulated mucosal immune response to gut-derived bacterial antigens is thought to be involved. Toll-like receptor ligands, especially lipopolysaccharide (LPS), contribute to the maintenance of the disease. It has previously been shown that the enzyme alkaline phosphatase (AP) is able to detoxify LPS, and the aim of this study was to examine a possible role in IBDs. METHODS: Intestinal AP (iAP) mRNA expression and LPS dephosphorylation in intestinal biopsies of control subjects and patients with IBD were examined, and the effect of orally administered iAP tablets on the progression of dextran sodium sulfate-induced colitis in rats was subsequently studied. RESULTS: In healthy persons, iAP mRNA and enzyme activity was high in the ileum relative to the colon. In patients with UC and CD, iAP mRNA expression was found to be markedly reduced when inflamed tissue was compared with non-inflamed tissue. Oral administration of iAP tablets to colitic rats resulted in a significant attenuation of colonic inflammation as reflected by reduced mRNA levels for tumour necrosis factor alpha, interleukin 1 beta, interleukin 6 and inducible nitric oxide synthase NOS (iNOS), a reduced iNOS staining and inflammatory cell influx, and a significantly improved morphology of the intestinal wall. CONCLUSIONS: The present study shows that epithelial iAP mRNA expression is reduced in patients with UC and CD. The rat model demonstrates that oral administration of active iAP enzymes in the intestinal tract results in a significant reduction of inflammation. This provides new insight on IBD pathology and a novel treatment approach to this severe inflammatory disease.

Eradication of established tumors by vaccination with Venezuelan equine encephalitis virus replicon particles delivering human papillomavirus 16 E7 RNA.

The etiological role of human papillomaviruses (HPV) in cervical and other cancers suggests that therapeutic vaccines directed against requisite viral antigens may eradicate tumors or their precursors. A Venezuelan equine encephalitis (VEE) alphavirus vector delivering the HPV16 E7 RNA was evaluated for antitumor efficacy using a murine E7+ tumor model. Vaccination with VEE replicon particles expressing E7 (E7-VRP) induced class I-restricted CD8+ T-cell responses as determined by IFN-gamma enzyme-linked immunospot (ELISPOT), tetramer, and cytotoxicity assays. E7-VRP vaccination prevented tumor development in all of the mice and effectively eliminated 7-day established tumors in 67% of tumor-bearing mice. The induction of protective T-cell responses was dependent on CD8+, but not CD4+ T cells. Long-lasting T-cell memory responses developed in E7-VRP-vaccinated mice as determined by complete protection from tumor challenge 3 months after the final vaccination. These promising results highlight the potent CD8+ T-cell-mediated antitumor effects elicited by VEE replicon-based vectors and support their further development toward clinical testing against cervical intraepithelial neoplasia or carcinoma.

Murine six-transmembrane epithelial antigen of the prostate, prostate stem cell antigen, and prostate-specific membrane antigen: prostate-specific cell-surface antigens highly expressed in prostate cancer of transgenic adenocarcinoma mouse prostate mice.

To identify genes that are differentially up-regulated in prostate cancer of transgenic adenocarcinoma mouse prostate (TRAMP) mice, we subtracted cDNA isolated from mouse kidney and spleen from cDNA isolated from TRAMP-C1 cells, a prostate tumor cell line derived from a TRAMP mouse. Using this strategy, cDNA clones that were homologous to human six-transmembrane epithelial antigen of the prostate (STEAP) and prostate stem cell antigen (PSCA) were isolated. Mouse STEAP (mSteap) is 80% homologous to human STEAP at both the nucleotide and amino acid levels and contains six potential membrane-spanning regions similar to human STEAP. Mouse PSCA (mPsca) shares 65% homology with human PSCA at the nucleotide and amino acid levels. mRNA expression of mSteap and mPsca is largely prostate-specific and highly detected in primary prostate tumors and metastases of TRAMP mice. Both mSteap and mPsca map to chromosome 5. Another known gene coding for mouse prostate-specific membrane antigen (mPsma) is also highly expressed in both primary and metastatic lesions of TRAMP mice. These results indicate that the TRAMP mouse model can be used to effectively identify genes homologous to human prostate-specific genes, thereby allowing for the investigation of their functional roles in prostate cancer. mSteap, mPsca, and mPsma constitute new tools for preventative and/or therapeutic vaccine construction and immune monitoring in the TRAMP mouse model that may provide insights into the treatment of human prostate cancer.

Human T cell responses to endogenously presented HLA-A*0201 restricted peptides of Simian virus 40 large T antigen.

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA-A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA-A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA-A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA-A*0201, Tag aa 396-405 and aa 577-585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396-405 and in T cell cultures of both donors Tag aa 577-585 specific T cells were initiated. The T cells against Tag aa 577-585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40.

Pharmacokinetic differences between a T cell-tolerizing and a T cell-activating peptide.

Vaccination with a peptide representing a CTL epitope from the human papillomavirus (HPV)16 E7 protein induces a specific CTL response that prevents the outgrowth of HPV16 E7-expressing tumors. In contrast, vaccination with a peptide encoding an adenovirus type 5 (Ad5) E1A CTL epitope results in CTL tolerance and enhanced growth of an Ad5 E1A-expressing tumor. It is unclear why these peptides induce such opposite effects. To determine whether a difference in pharmacokinetics can explain the functional contrasts, tritiated Ad5 E1A and HPV16 E7 peptides were injected into mice. Results show that the tolerizing peptide spread through the body 16 times faster than the activating peptide and was cleared at least 2 times faster. The HPV16 E7 peptide kinetics correlated with the kinetics of HPV16 E7-specific CTL induction. In contrast, Ad5 E1A peptide injection resulted in physical deletion of preexisting Ad5 E1A-specific CTLs within 24 h after injection. This tolerization occurred at the time when the peptide reached its maximum peptide concentration in the organs. These data suggest that ubiquitous expression of the tolerizing Ad5 E1A peptide within a short period of time causes activation-induced cell death of Ad5 E1A-specific CTLs. Therefore, information on the pharmacokinetics of peptides is vital for the safety and efficacy of peptide-based vaccines.

Prospect for immunotherapy of acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is diagnosed in approximately 100000 people worldwide per year and 70% of the patients are children. Most children have a good prognosis, as almost 80% will be cured, however only 30% of adults are cured. Additionally, the current chemotherapies have long-lasting and severe side-effects. These findings indicate that the search for better and safer treatment modalities for ALL is still important. As leukemia directly affects the human immune cells, immunotherapeutic approaches have long been ignored as treatment options for this disease. However, increased knowledge of the immune system has opened new opportunities for immune modulation that could be of benefit to leukemia patients. Several recent advances towards immunotherapy of ALL will be discussed.

A murine model for the effects of pelvic radiation and cisplatin chemotherapy on human papillomavirus vaccine efficacy.

Therapeutic human papillomavirus (HPV) vaccines for cervical cancer depend on a competent immune system to be effective. However, cancer patients are often found to be immunosuppressed, which could be attributable to prior radiation, chemotherapy, or the tumor burden itself. This study investigated whether pelvic radiation or cisplatin treatment affected the efficacy of an HPV vaccine and how long these effects lasted. Mice were given pelvic radiation, 2 Gy/day to a total dose of 45 Gy, or 5 mg/kg/week of cisplatin for 3 weeks. Mice were then immunized with an HPV-16 peptide vaccine between 0 and 16 weeks after their treatment. An ELISPOT analysis revealed that a reduced level of peptide-specific, IFNgamma-producing spleen cells was present in immunized mice treated previously with pelvic radiation or cisplatin compared with immunized mice that had not been treated. However, when mice were challenged with HPV-16-expressing tumor cells, immunized mice developed no tumors, regardless of prior treatment, whereas nonimmunized mice did develop tumors. Our results suggest that pretreatment with pelvic radiation or cisplatin alone does not prevent the induction of an effective immune response by a peptide vaccine. These data will have important implications for immunotherapeutic treatment of pretreated cancer patients, especially in the adjuvant setting when immunosuppression by tumor burden would be low.

Defined flanking spacers and enhanced proteolysis is essential for eradication of established tumors by an epitope string DNA vaccine.

Loss of immunogenic epitopes by tumors has urged the development of vaccines against multiple epitopes. Recombinant DNA technologies have opened the possibility to develop multiepitope vaccines in a relatively rapid and efficient way. We have constructed four naked DNA-based multiepitope vaccines, containing CTL, Th cell, and B cell epitopes of the human papillomavirus type 16. Here we show that gene gun-mediated vaccination with an epitope-based DNA vaccine protects 100% of the vaccinated mice against a lethal tumor challenge. The addition of spacers between the epitopes was crucial for the epitope-induced tumor protection, as the same DNA construct without spacers was significantly less effective and only protected 50% of the mice. When tested for therapeutic potential, only the epitope construct with defined spacers significantly reduced the size of established tumors, but failed to induce tumor regression. Only after targeting the vaccine-encoded protein to the protein degradation pathway by linking it to ubiquitin, the vaccine-induced T cell-mediated eradication of 100% of 7-day established tumors in mice. The finding that defined flanking sequences around epitopes and protein targeting dramatically increased the efficacy of epitope string DNA vaccines against established tumors will be of importance for the further development of multiepitope DNA vaccines toward clinical application.

Mechanism of immune dysfunction in cancer mediated by immature Gr-1+ myeloid cells.

The mechanism of tumor-associated T cell dysfunction remains an unresolved problem of tumor immunology. Development of T cell defects in tumor-bearing hosts are often associated with increased production of immature myeloid cells. In tumor-bearing mice, these immature myeloid cells are represented by a population of Gr-1(+) cells. In this study we investigated an effect of these cells on T cell function. Gr-1(+) cells were isolated from MethA sarcoma or C3 tumor-bearing mice using cell sorting. These Gr-1(+) cells expressed myeloid cell marker CD11b and MHC class I molecules, but they lacked expression of MHC class II molecules. Tumor-induced Gr-1(+) cells did not affect T cell responses to Con A and to a peptide presented by MHC class II. In sharp contrast, Gr-1(+) cells completely blocked T cell response to a peptide presented by MHC class I in vitro and in vivo. Block of the specific MHC class I molecules on the surface of Gr-1(+) cells completely abrogated the observed effects of these cells. Thus, immature myeloid cells specifically inhibited CD8-mediated Ag-specific T cell response, but not CD4-mediated T cell response. Differentiation of Gr-1(+) cells in the presence of growth factors and all-trans retinoic acid completely eliminated inhibitory potential of these cells. This may suggest a new approach to cancer treatment.

Physical interaction of human papillomavirus virus-like particles with immune cells.

Human papillomavirus virus-like particles (HPV VLP) and chimeric VLP are immunogens that are able to elicit potent anti-viral/tumor B and T cell responses. To investigate the immunogenicity of VLP, we determined which cells of the immune system are able to bind HPV-16 VLP. VLP were found to bind very well to human and mouse immune cells that expressed markers of antigen-presenting cells (APC) such as MHC class II, CD80 and CD86, including dendritic cells, macrophages and B cells. mAb blocking studies identified Fc gamma RIII (CD16) as one of the molecules to which the VLP can bind both on immune cells and foreskin epithelium. However, transfection of a CD16(-) cell line with CD16 did not confer binding of VLP. Splenocytes from Fc gamma RIII knockout mice showed a 33% decrease in VLP binding overall and specifically to subsets of APC. These combined data support a role for CD16 as an accessory molecule in an HPV VLP-receptor complex, possibly contributing to the immunogenicity of HPV VLP.

Cervical cancer vaccines: emerging concepts and developments.

Certain human cancers are linked to infection by oncogenic viruses that are able to cause transformation of the normal host cell into a cancerous cell. Human papillomavirus (HPV) DNA and expression of viral transforming proteins are found in virtually all cervical cancer cells, indicating an important role of this virus in the pathogenesis of the disease. Evidence exists that the immune response to cancer cells can play a major role in determining the outcome of disease. The fact that HPV is a necessary cause for cervical cancer provides a clear opportunity to develop a therapeutic vaccine against the virus to treat patients with cervical cancer at its early and late stages. Development of a prophylactic vaccine for HPV would also reduce the incidence of cervical neoplasias by preventing virus infection. Various candidate HPV vaccines are being developed and tested in animal models and/or in human clinical trials. These HPV vaccines, both preventive and therapeutic, are the subjects of this review.

Molecular characterization of melanoma-derived antigens.

In the last decade, many antigens expressed by tumors and recognized by the immune system have been identified. Melanoma was among the first tumors found to express such tumor-associated antigens, and, therefore, melanoma is currently one of the best and extensively studied tumors for which new techniques have been introduced to optimize the characterization of tumor antigens. In this chapter, we discuss the techniques used for identification of melanoma-expressed antigens recognized by cytotoxic T-lymphocytes (CTLs). In more detail, we describe in Subheading 3. the reverse immunology method.

Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles.

It has been postulated that upon binding to a cell surface receptor, papilloma virus-like particles (VLPs) gain entry into the cytosol of infected cells and the capsid proteins L1 and L2 can be processed in the MHC class I presentation pathway. Vaccination of mice with human papilloma virus-like particles consisting of capsid proteins L1 and L2 induced a CD8-mediated and perforin dependent protective immune response against a tumor challenge with human papilloma virus transformed tumor cells, which express only minute amounts of L1 protein. Here we show that HPV16 capsid proteins stimulate a MHC class I restricted CTL response with human peripheral blood lymphocytes (PBL) in vitro. The vigorous response was specific for VLP-infected target cells and was MHC class I restricted. Moreover we show the presence of at least one HLA-A*0201 restricted CTL epitope within the HPV-16 capsid proteins by using a VLP-'infected' HLA-A*0201 transfected human cell line as target cells. These results demonstrated that VLPs can induce a HPV16 capsid protein-specific immune response in humans, allowing the monitoring of immune responses induced by vaccines based on chimeric VLPs carrying additional immunogenic peptides or proteins in therapeutical applications in human patients.

Chimeric papillomavirus virus-like particles induce a murine self-antigen-specific protective and therapeutic antitumor immune response.

The use of chimeric virus-like particles represents a new strategy for delivering tumor antigens to the immune system for the initiation of antitumor immune responses. Immunization of DBA/2 mice with the P1A peptide derived from the P815 tumor-associated antigen P1A induced specific T-cell tolerance, resulting in progression of a regressor P815 cell line in all animals. However, immunization with a human papillomavirus type 16 L1 virus-like particle containing the P1A peptide in the absence of adjuvant induced a protective immune response in mice against a lethal tumor challenge with a progressor P815 tumor cell line. Additionally, we demonstrated that these chimeric virus-like particles could be used therapeutically to suppress the growth of established tumors, resulting in a significant survival advantage for chimeric virus-like particle-treated mice compared with untreated control mice. Chimeric virus-like particles can thus be used as a universal delivery vehicle for both tolerizing and antigenic peptides to induce a strong protective and therapeutic antigen-specific antitumor immune response.

Modulation of the immune response and tumor growth by activated Ras.

As a result of its transforming abilities, activated Ras is expressed in a great number of cancers. The ras mutation frequency varies between 95% in pancreatic cancer and 5% in breast cancer. In leukemia, the highest frequency (30%) is found in acute myeloid leukemia. The presence of ras mutations has been correlated with a poor prognosis and negative clinical outcome. This suggests that mutated Ras activates mechanisms, which favor tumor growth, enhance the metastatic capacity of tumors or modulate tumor-specific immune responses. Several new functions of Ras, such as downregulation of major histocompatibility complex molecules, upregulation of certain cytokines, growth factors and degradative enzymes have been uncovered in the last decade. Additionally, mutated Ras can also serve as a primary target for the development of immunotherapy or drug therapy. This review will discuss the mechanisms by which Ras expressing tumors are able to evade destruction by the immune system and enhance their growth and metastatic potential. It will further elaborate on the attempts to develop successful immunotherapy and drug therapy targeting Ras expressing tumors.

Papillomavirus virus-like particles as anticancer vaccines.

Papillomavirus virus-like particles (VLPs) are empty, non-replicative, non-infectious particles that retain conformationally correct epitopes for the generation of antibody responses to the viral capsid proteins. Chimeric human papillomavirus (HPV) virus-like particles incorporating non-structural virus proteins offer an exciting approach for combined prophylactic and therapeutic vaccines against HPV-induced lesions. Both HPV VLPs and chimeric VLPs can induce potent humoral and cellular immune responses when injected into mice, leading to the generation of virus-neutralizing antibodies, priming of CD8+ T-cells and activation of cytotoxic T-cell effector functions. This review summarizes recent advances in the production of chimeric VLPs, the immune response elicited by VLPs and chimeric VLPs, and their ability to generate strong protective and therapeutic antitumor immune responses.

Active immunization against cancer cells: impediments and advances.

Immunotherapy of cancer is still mainly an experimental treatment. Some monoclonal antibodies have been approved for adjuvant therapy of cancer in patients, but active immunization strategies have not yet matured to this stage. The fact that vaccination against viral diseases is effective has primed high expectations for successful vaccination against cancer as well. Indeed, in some animal models, therapeutic results could be obtained against short-term established tumors, which paved the way for clinical trials. However, the first results with active immunization in cancer patients were disappointing and this led to a careful examination of current protocols and the search for more effective approaches. Evaluation of the available data suggests that cancer patients may not be comparable in their immune response to cancer vaccines with healthy persons. Furthermore, the tumor seems to be able to develop several immune-escape mechanisms, which either inactivate the specific immune cells or prevent activation of potential effector mechanisms against the tumor. Here, we review the impediments that have been identified in murine models and clinical trials for immunotherapy of cancer. It will be important to study the hurdles to come to a better understanding of the immune evasion of tumors and to achieve efficient activation of the immune system in cancer patients against the tumor. This knowledge will open new possibilities for active immunization against cancer.

Cellular immunity and immunotherapy against deoxyribonucleic acid virus-induced tumors.

An important role in the immune defense against deoxyribonucleic acid virus induced tumors is mediated by T-cells, as is evident from aetiological, animal model, and clinical data. In this review the most recent observations in this field are described for three prominent members of this family of viruses, namely human papillomavirus associated with human cervical cancer, human adenovirus associated with lung infections in humans and tumors in rodents, and simian virus 40 associated with rodent tumors and human mesothelioma, osteosarcoma and ependymoma.

The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas.

Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism through which tumour cells, upon treatment with anti-tumour MAbs, are eliminated in vivo. However, the relative importance of various parameters that influence the efficacy of ADCC is unclear. Here we present in vitro data on the impact of MAb affinity and antigen density on ADCC, as obtained by comparison of two MAbs against the tumour-associated antigen Ep-CAM. The low-affinity MAb 17-1A (Ka = 5 x 10(7)M(-1)) currently used for therapy, and the high-affinity MAb 323/A3 (Ka = 2 x 10(9) M(-1)), were compared in ADCC experiments against murine and human tumour target cells transfected with the Ep-CAM cDNA under the control of an inducible promoter to enable regulation of the target antigen expression levels. Data obtained from these studies revealed that the high-affinity MAb, in contrast to the low-affinity MAb, could mediate killing of tumour cells with low antigen expression levels. Even at comparable MAb-binding levels, ADCC mediated by the high-affinity MAb was more effective. The kinetics of ADCC was also found to be determined by the level of antigen expression, and by the affinity and the concentration of the MAb used. The efficacy of ADCC with both low- and high-affinity MAbs further depended on adhesive interactions between effector and target cells mediated by CD18. However, at every given MAb concentration these interactions were of less importance for the high-affinity MAb than for the low-affinity MAb. As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease.

PBLs of early breast carcinoma patients with a high nuclear grade tumor unlike PBLs of cervical carcinoma patients do not show a decreased TCR zeta expression but are functionally impaired.

Peripheral blood lymphocytes (PBLs) of patients with cervical intraepithelial neoplasia (CIN), cervical carcinoma, or early breast carcinoma were tested for the expression of T cell receptor zeta chain (TCR zeta) and CD16 zeta chain and production of interferon-gamma (IFN gamma) and interleukin (IL) 10. We found that in all patients with CIN and invasive cervical carcinoma, PBLs showed a reduced TCR zeta and CD16 zeta expression and a significant down-regulation in IFN gamma production (a T helper 1 cytokine) after anti-CD3 stimulation. However, the IL 10 secretion (a T helper 2 cytokine) was not diminished after anti-CD3 stimulation. This indicates that only T helper 1 cells are affected by the down-regulation of the TCR zeta chain expression. We also analyzed PBLs of 12 patients with early breast carcinoma. In these patients, we found TCR zeta and CD16 zeta expression down-regulation in 2 of 12 patients. Six of 12 patients had an enhanced TCR zeta expression. The enhanced TCR zeta expression correlated with a reduced IFN gamma expression after anti-CD3 stimulation. These data show that in general PBLs of early breast carcinoma patients, unlike those of cervical carcinoma patients, do not show a decreased TCR zeta expression. However, a functional impairment of T cells was observed in the subgroup of early breast carcinoma patients with a high nuclear grade of their tumor.