A novel therapeutic vaccine composed of a rearranged human papillomavirus type 16 E6/E7 fusion protein and Fms-like tyrosine kinase-3 ligand induces CD8+ T cell responses and antitumor effect.

The development of cervical cancer is mainly caused by infection with high risk genotypes of human papillomavirus, particularly type 16 (HPV16), which accounts for more than 50% of cervical cancer. The two early viral oncogenes, E6 and E7, are continuously expressed in cervical cancer cells and are necessary to maintain the malignant cellular phenotype, thus providing ideal targets for immunotherapy of cervical cancer. In this study, a novel vaccine strategy was developed based on a rationally shuffled HPV16 E6/E7 fusion protein, the addition of Fms-like tyrosine kinase-3 ligand (Flt3L) or the N domain of calreticulin (NCRT), and the usage of a CpG adjuvant. Four recombinant proteins were constructed: m16E6E7 (mutant E6/E7 fusion protein), rm16E6E7 (rearranged mutant HPV16 E6/E7 fusion protein), Flt3L-RM16 (Flt3L fused to rm16E6E7), and NCRT-RM16 (NCRT fused to rm16E6E7). Our results suggest that Flt3L-RM16 was the most potent of these proteins in terms of inducing E6- and E7-specific CD8+ T cell responses. Additionally, Flt3L-RM16 significantly induced regression of established E6/E7-expressing TC-1 tumors. Higher doses of Flt3L-RM16 trended toward higher levels of antitumor activity, but these differences did not reach statistical significance. In summary, this study found that Flt3L-RM16 fusion protein is a promising therapeutic vaccine for immunotherapy of HPV16-associated cervical cancer.

Role of the K(Ca)3.1 K+ channel in auricular lymph node CD4+ T-lymphocyte function of the delayed-type hypersensitivity model.

BACKGROUND AND PURPOSE: The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of K(Ca)3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice. EXPERIMENTAL APPROACH: The expression patterns of K(Ca)3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized, but non-challenged (Ox+/-) and Ox-sensitized and -challenged (Ox+/+)] using real-time polymerase chain reaction, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of K(Ca)3.1 blockade were examined by the administration of selective K(Ca)3.1 blockers. KEY RESULTS: Significant up-regulation of K(Ca)3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, K(Ca)3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor (REST) was significantly down-regulated. Pharmacological blockade of K(Ca)3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0/G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the K(Ca)3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+. CONCLUSIONS AND IMPLICATIONS: The up-regulation of K(Ca)3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice; and K(Ca)3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.

Synthetic vaccine for the treatment of lesions caused by high risk human papilloma virus.

Until recently, therapeutic cancer vaccines only sporadically led to long-term clinical responses. We here report on a novel vaccine modality, characterized by the administration of long (23-45 amino acids) synthetic peptides in incomplete Freund adjuvant (mineral oil-based, Montanide ISA-51), delivered subcutaneously. Such vaccines were first demonstrated to be much more potent in preclinical T-cell response induction and tumor therapy experiments than short major histocompatibility complex class I-binding peptides that have been used extensively in the clinic. A long-peptide vaccine consisting of 13 overlapping peptides, together covering the entire length of the 2 oncogenic proteins E6 and E7 of high-risk human papilloma virus type 16 (HPV16), caused complete regression of all lesions and eradication of virus in 9 of 20 women with high-grade vulvar intraepithelial neoplasia. The nature and strength of the vaccine-induced T-cell response were significantly correlated with the clinical response. This vaccine promises to be of use, not only in patients with premalignant lesions caused by high-risk HPV16, but also in patients with malignant tumors caused by this virus, including HPV16-positive cervical cancer, anal cancer, and head and neck cancer.

Downregulation of Ches1 and other novel genes in oral cancer cells chronically exposed to areca nut extract.

BACKGROUND: This study was undertaken to identify the genes in response to areca nut extract, a potential carcinogen of oral cancer. METHODS: Two oral cancer sublines chronically treated with areca nut extract were established. Methods such as microarray and immunohistochemistry were used to screen and validate the genes' altered expressions in areca nut extract-sublines or in cancer tissues. RESULTS: A total of 35 genes were differentially expressed in both sublines. Several functional pathways were significantly altered. Six genes were confirmed over 2-fold of changes, including Ches1. Functional analyses showed that overexpression of Ches1 suppressed cell growth and arrested cells in the G2/M phase. Consistently, this gene has reduced expression in 52% of oral cancer tissues, which was significantly correlated with the areca nut chewing habit of patients (p = .04). CONCLUSION: We identified 35 candidates and validated 6 genes that may be associated with areca nut-induced oral cancer. Loss of Ches1 may be attributed to areca nut extract-induced oral carcinogenesis.

Peroxisome proliferator-activated receptor gamma ligands enhance human B cell antibody production and differentiation.

Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)gamma/RXRalpha pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPARgamma. We also show that nanomolar levels of natural (15-deoxy-Delta(12,14)-prostaglandin J(2)) or synthetic (rosiglitazone) PPARgamma ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPARgamma antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPARgamma and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXRalpha ligand (9-cis-retinoic acid) and PPARgamma ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPARgamma ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPARgamma ligands. These new findings indicate that low doses of PPARgamma/RXRalpha ligands could be used as a new type of adjuvant to stimulate Ab production.

Enhancement of DNA vaccine potency through coadministration of CIITA DNA with DNA vaccines via gene gun.

Administration of DNA vaccines via gene gun has emerged as an important form of Ag-specific immunotherapy. The MHC CIITA is a master regulator of MHC class II expression and also induces expression of class I molecules. We reasoned that the gene gun administration of CIITA DNA with DNA vaccines employing different strategies to improve MHC I and II processing could enhance DNA vaccine potency. We observed that DC-1 cells transfected with CIITA DNA lead to higher expression of MHC I and II molecules, leading to enhanced Ag presentation through the MHC I/II pathways. Furthermore, our data suggested that coadministration of DNA-encoding calreticulin (CRT) linked to human papillomavirus (HPV) 16 E6 Ag (CRT/E6) with CIITA DNA leads to enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. In addition, coadministration of the combination of CRT/E6 DNA with CIITA DNA and DNA encoding the invariant chain (Ii) linked to the pan HLA-DR-reactive epitope (Ii-PADRE) further enhanced E6-specific CD8(+) T cell immune responses in vaccinated mice. Treatment with the combination vaccine was also shown to enhance the antitumor effects and to prolong survival in TC-1 tumor-bearing mice. Vaccination with the combination vaccine also led to enhanced E6-specific CD8(+) memory T cells and to long-term protection against TC-1 tumors and prolonged survival in vaccinated mice. Thus, our findings suggest that the combination of CIITA DNA with CRT/E6 and Ii-PADRE DNA vaccines represents a potentially effective means to combat tumors in the clinical setting.

A cardiovascular drug rescues mice from lethal sepsis by selectively attenuating a late-acting proinflammatory mediator, high mobility group box 1.

The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF, IL-1, and IFN-gamma) and late (e.g., high mobility group box 1 (HMGB1) protein) proinflammatory cytokines. The recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted investigation for development of new experimental therapeutics. We found that many steroidal drugs (such as dexamethasone and cortisone) and nonsteroidal anti-inflammatory drugs (such as aspirin, ibuprofen, and indomethacin) failed to influence endotoxin-induced HMGB1 release even at superpharmacological concentrations (up to 10-25 microM). However, several steroid-like pigments (tanshinone I, tanshinone IIA, and cryptotanshinone) of a popular Chinese herb, Danshen (Salvia miltiorrhiza), dose dependently attenuated endotoxin-induced HMGB1 release in macrophage/monocyte cultures. A water-soluble tanshinone IIA sodium sulfonate derivative (TSNIIA-SS), which has been widely used as a Chinese medicine for patients with cardiovascular disorders, selectively abrogated endotoxin-induced HMGB1 cytoplasmic translocation and release in a glucocorticoid receptor-independent manner. Administration of TSNIIA-SS significantly protected mice against lethal endotoxemia and rescued mice from lethal sepsis even when the first dose was given 24 h after the onset of sepsis. The therapeutic effects were partly attributable to attenuation of systemic accumulation of HMGB1 (but not TNF and NO) and improvement of cardiovascular physiologic parameters (e.g., decrease in total peripheral vascular resistance and increase in cardiac stroke volume) in septic animals. Taken together, these data re-enforce the pathogenic role of HMGB1 in lethal sepsis, and support a therapeutic potential for TSNIIA-SS in the treatment of human sepsis.

ISCOMATRIX adjuvant: an adjuvant suitable for use in anticancer vaccines.

Human Papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are associated with cervical cancer development and progression and can therefore be used as target antigens for cancer immunotherapy. In this study we evaluated the immunogenicity in mice, of different vaccine formulations using recombinant HPV16 derived E6E7 or E7GST fusion proteins. When co-administered with ISCOMATRIX adjuvant, these E6E7 proteins consistently induced E7 specific CTL, in vivo tumor protection, antibody and DTH responses. ISCOMATRIX adjuvant has been developed for use in the formulation of novel human vaccines and has been evaluated for safety and toxicity in human trials. A formulation containing aluminum hydroxide (Al(OH)3) gave a lesser degree of E7 specific antibody, and no local E7 specific CTL response but similar DTH and tumor protection. These findings demonstrate the potential of ISCOMATRIX adjuvant to stimulate both cellular and humoral immune responses to endogenously processed target antigens, and hence is the preferred adjuvant when CTL responses are desirable.

The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p.

The JC12 monoclonal antibody recognizes a previously unknown nuclear protein that showed a restricted distribution in normal tonsil and was also overexpressed in a subset of diffuse large B-cell lymphomas. Using this reagent, we expression cloned cDNAs encoding its antigenic target and identified this protein as a novel putative transcription factor, FOXP1. The FOXP1 protein sequence contains predicted domains characteristic of transcription factors, including a winged helix DNA-binding motif, a second potential DNA-binding motif, a C(2)H(2) zinc finger, nuclear localization signals, coiled-coil regions, PEST sequences, and potential transactivation domains. The FOXP1 gene has been mapped to chromosome 3p14.1, a region that commonly shows loss of heterozygosity in a wide range of tumors and which is reported to contain a tumor suppressor gene(s). Using tissue arrays and immunohistochemistry, we demonstrate that both the FOXP1 mRNA and protein are widely expressed in normal tissues. The levels of FOXP1 mRNA were compared in paired normal and tumor tissues (from the same patient) using a tissue array containing cDNAs extracted from 68 samples taken from kidney, breast, prostate, uterus, ovary, cervix, colon, lung, stomach, rectum, small intestine, and from nine cancer cell lines. Differences in FOXP1 mRNA expression between normal and tumor samples were observed in 51% of cases. Most striking was the comparative loss of expression in 73% of colon tumors and comparative overexpression of FOXP1 mRNA in 75% of stomach tumors. Analysis of the FOXP1 mRNA expression in normal tissues (not taken from cancer patients) indicated that loss of FOXP1 expression may occur in some histologically normal tissues adjacent to tumors. Immunohistochemical analysis of FOXP1 protein expression was performed on 128 solid tumors, including 16 renal, 9 breast, 12 lung, 20 colon, 21 stomach, 10 head and neck, 35 prostate, and 5 pancreatic cases. Complete loss of expression, increased expression, and cytoplasmic mislocalization of the predominantly nuclear FOXP1 protein were frequently observed in neoplastic cells. Our study identifies FOXP1 as a new candidate tumor suppressor gene localized to the chromosome 3p14.1 region.

Syk tyrosine kinase and B cell antigen receptor (BCR) immunoglobulin-alpha subunit determine BCR-mediated major histocompatibility complex class II-restricted antigen presentation.

Stimulation of CD4(+) helper T lymphocytes by antigen-presenting cells requires the degradation of exogenous antigens into antigenic peptides which associate with major histocompatibility complex (MHC) class II molecules in endosomal or lysosomal compartments. B lymphocytes mediate efficient antigen presentation first by capturing soluble antigens through clonally distributed antigen receptors (BCRs), composed of membrane immunoglobulin (Ig) associated with Ig-alpha/Ig-beta heterodimers which, second, target antigens to MHC class II-containing compartments. We report that antigen internalization and antigen targeting through the BCR or its Ig-alpha-associated subunit to newly synthesized class II lead to the presentation of a large spectrum of T cell epitopes, including some cryptic T cell epitopes. To further characterize the intracellular mechanisms of BCR-mediated antigen presentation, we used two complementary experimental approaches: mutational analysis of the Ig-alpha cytoplasmic tail, and overexpression in B cells of dominant negative syk mutants. Thus, we found that the syk tyrosine kinase, an effector of the BCR signal transduction pathway, is involved in the presentation of peptide- MHC class II complexes through antigen targeting by BCR subunits.