Cooperative effects of Mycobacterium tuberculosis Ag38 gene transduction and interleukin 12 in vaccination against spontaneous tumor development in proto-neu transgenic mice.

An approach to stimulating an immune response against tumors is to transduce tumor cells with bacterial genes, which represent a "danger signal" and can induce a wide immune response. Mycobacterium tuberculosis genes and their encoded proteins play a pivotal role in linking innate and cell-mediated adaptive immunity and represent ideal candidates as immune adjuvants for tumor vaccines. The efficacy of a cancer vaccine, obtained by transduction of a mammary tumor cell line with the M. tuberculosis Ag38 gene, was investigated in female mice transgenically expressing the rat HER-2/neu proto-oncogene. These mice spontaneously develop stochastic mammary tumors after a long latency period. The onset of spontaneous mammary tumors was significantly delayed in mice vaccinated with Ag38-transduced cells but not in mice vaccinated with nontransduced cells as compared with untreated mice. Protection from spontaneous tumor development was increased when mice were vaccinated with the mycobacterium gene-transduced vaccine plus a systemic administration of interleukin 12 (IL-12) at a low dose. Mice vaccinated with nontransduced cells plus IL-12 developed tumors, with only a slight delay in tumor appearance as compared with the control group. Lymphocytes obtained from lymph nodes of mice vaccinated with transduced cells secreted high levels of IFN-gamma. CD3+CD8+ spleen cells derived from these mice responded to the tumor with IFN-gamma production. These data indicate the efficacy of a short-term protocol of vaccinations exploiting the adjuvant potency of a M. tuberculosis gene and low doses of IL-12 in a model of stochastic development of mammary tumors. This adjuvant approach may represent a promising immunotherapeutic strategy for cancer immunization.

Evaluation of the balance between angiogenic and antiangiogenic circulating factors in patients with breast and gastrointestinal cancers.

Angiogenesis is a critical determinant of tumor growth. Tumor cells produce or induce angiogenic molecules that act specifically on endothelial cells (ECs) but also release angiostatic molecules. Thus, tumor angiogenesis represents a net balance between positive and negative regulators of neovascularization. Sera from patients with breast or gastrointestinal cancers were evaluated for their capacity to selectively modulate the proliferation of human umbilical vein ECs; sera from 15 of 78 (19%) breast cancer patients and 8 of 53 (15%) gastrointestinal cancer patients induced human umbilical vein EC growth, whereas sera from 4 of 78 (5%) breast cancer patients and 1 of 53 (2%) gastrointestinal cancer patients inhibited EC proliferation. Growth-stimulatory sera were significantly more frequent among postmenopausal (14 of 53) than premenopausal (1 of 25) breast cancer patients; inhibitory activity was observed in 3 of 25 premenopausal patients versus 1 of 53 postmenopausal individuals. The half-life of serum-stimulating and -inhibiting factors seemed to differ, because stimulatory activity but not inhibitory activity was decreased at 5 days after surgery. The levels of vascular endothelial growth factor were elevated in about 45% of patients with growth-stimulatory sera, whereas the serum inhibition of EC growth was found to be due, at least in part, to high levels of soluble thrombospondin.

Innate immunity in breast carcinoma.

The innate immune response, which depends on so-called pattern-recognition receptors (PRRs) is an evolutionarily old immune response able to elicit a defensive response against a vast array of pathogens. The purpose of this review is to revisit the role of innate immunity in breast carcinoma from the oldest therapeutic approach using bacillus Calmette-Guerin to the recent findings on the manipulation of the PRR pathways with unmethylated cytosine-guanosine dinucleotides (CpG motifs). Encouraging results have been obtained in prevention and local treatment of murine mammary tumors using tumor cells engineered to express stably mycobacterial antigens or directly using CpG-containing oligonucleotides. The experimental findings raise the possibility of successful anti-tumor management through stimulation of innate immunity in women at high risk of developing breast cancer and in breast cancer patients with reasonable immunological performance and low tumor load.

Combination of a CpG-oligodeoxynucleotide and a topoisomerase I inhibitor in the therapy of human tumour xenografts.

The study was conducted to investigate the effects of a novel therapeutic approach, i.e. the combination of chemotherapy and immunotherapy, against a human prostate carcinoma xenograft. A topoisomerase I inhibitor, topotecan, and CpG-containing oligodeoxynucleotides (CpG-ODN) were combined. Athymic mice bearing the PC-3 human prostate carcinoma were treated with the maximum tolerated dose (MTD) of topotecan (3 weekly treatments) and with repeated treatments of CpG-ODN (40 and 20 microg/mouse); tumour growth and lethal toxicity were monitored. Topotecan effect on CpG-ODN-induced production of interleukin (IL) 12, interferon (IFN)-gamma and tumour necrosis factor-alpha was also assessed. Since topotecan pretreatment differentially influenced CpG-ODN-induced production of IL-12 and IFN-gamma, the antitumour effects of the two therapies were investigated in a sequential (full topotecan regimen followed by CpG-ODN) or in an alternating sequence (starting with CpG-ODN). Topotecan inhibited PC-3 tumour growth, inducing 95% tumour volume inhibition. All combined treatments resulted in a significant delay in tumour growth, compared to the effects in topotecan-treated mice (P<0.01, by analysis of tumour growth curves). The combination regimens were well tolerated, except for the alternating sequence of 40 microg CpG-ODN and topotecan, which resulted in three out of eight toxic deaths. This alternating sequence was highly toxic even when another cytotoxic drug (doxorubicin) was used in healthy mice. In conclusion, the combination of topotecan and CpG-ODN increased antitumour effects over chemotherapy alone in the growth of a human prostate carcinoma xenograft. Administration sequence was critical to the combination toxicity: the complete regimen of the cytotoxic drug followed by repeated administrations of the immunomodulator seemed the most promising for further investigations.

Antibody response after vaccination with antigen-pulsed dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system capable of initiating immune responses to antigens. It is also well documented that cancer patients often experience anergy against tumor antigens. In this study we selected the best protocol for inducing the production of antibodies against the HER2 oncoprotein using DCs to overcome anergy. Murine DCs were pulsed in vitro, using different protocols, with recombinant HER2 fused to a human Fc (in order to improve DC antigen uptake) and were used to vaccinate mice. The obtained results indicate that antigen-pulsed DCs can induce an antibody response and that adding CpG after antigen pulsing greatly increases anti-HER2 antibody production.

High level antibody response to retrovirus-associated but not to melanocyte lineage-specific antigens in mice protected against B16 melanoma.

Mice vaccinated with Mycobacterium tuberculosis Ag38 gene-transduced B16 melanoma cells showed significant protection from intravenous challenge with parental B16 melanoma cells. No cytotoxic T-cell activity was found against melanoma cells, although the endogenous presence of the mycobacterial gene induced a preferential Th1 response. After immunization, a low serological response against melanoma cells was detected, while a high titer of antibodies directed to parental B16 cells, mainly of IgG2(a) isotype, was found in protected mice after challenge. These antibodies exhibited complement-dependent cytotoxicity against melanoma cells in vitro, while in vivo, used in passive immunization, they induced a decrease in a number of experimental B16 lung metastases. Most of the antibodies were directed against endogenous murine leukemia viruses. No reactivity against melanocyte lineage-specific antigens was observed. In particular, no reactivity was found in sera from protected mice against tyrosinase-related protein 2 (TRP-2), either stably expressed in a non-melanoma cell line or obtained by in vitro transcription-translation, or against tyrosinase, TRP-1 and gp100 antigens immunoprecipitated from B16 cells. Thus, in the B16 murine model, the presence of dominant viral antigens induces a very strong humoral response that might be protective and may inhibit or mask the presence of minor clonotypes.

Topical administration of a doxorubicin-specific monoclonal antibody prevents drug-induced mouth apoptosis in mice.

One of the most severe side effects of anti-tumour chemotherapy is mucositis due to drug toxicity for rapidly dividing cells. We show here that anti-DXR monoclonal antibodies can prevent DXR-induced damage. Indeed, apoptosis, confined to the proliferative compartment of the basal mucosa, observed in the tongue of DXR-treated mice was completely inhibited by topical application of the anti-DXR antibodies.

Apoptosis of hair follicle cells during doxorubicin-induced alopecia in rats.

Alopecia is a common side effect of cancer chemotherapy, and to date, little progress has been made in alopecia prevention or treatment. The present studies were undertaken to topographically localize the site of injury in the hair follicle after doxorubicin (DXR) administration and to investigate the mechanism of DXR-induced alopecia. Tissue samples of head and proximal neck skin obtained from newborn rats treated with DXR were histologically examined by light and electron microscopy and stained by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Light microscopy revealed pyknotic cells in the matrix and in the upper bulb and a decrease in the number of mitotic cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling analysis evidenced cells with nuclear staining indicative of apoptosis, as confirmed by ultrastructural analysis. Kinetics studies indicated that even a single DXR treatment induced apoptosis and a decrease in mitotically active cells. Our data show that DXR treatment induces injury in a cell subset localized in the hair matrix. The successful prevention of drug-induced alopecia in patients may depend on the selective protection of these cells of the hair follicle.

Segregation of type 1 cytokine production in human peripheral blood lymphocytes: phenotypic differences between IFN-gamma and IL-2-producing cells in the CD8+ T cell subset.

T cell clones are classified as type 0, 1 or 2 depending on the lymphokines they produce. However, it has remained unclear whether single cells of a given type produce one or several cytokine species. Flow cytometric analysis of peripheral blood lymphocytes (PBL) obtained from 20 healthy donors for the production of the type 1 cytokines IFN-gamma and IL-2 revealed very few cells that co-expressed both cytokines independently of the mitogenic stimulus used for PBL activation. Similarly, kinetic studies of cytokine synthesis indicated a low percentage of IFN-gamma/IL-2 double-positive T cells at all time points. Reverse transcription-PCR analysis of sorted IL-2- and IFN-gamma-positive T cells showed the presence of IL-2- or IFN-gamma-specific mRNA only in those cells expressing the corresponding cytokine. This segregation of the two type 1 cytokines was lost in long-term cultured T cells and in T cell clones. A high percentage of cells expressing only IL-2 or IFN-gamma was observed even when the production of these cytokines was evaluated on CD4- and CD8+ subsets. Moreover, in some healthy individuals, IFN-gamma and IL-2 production by CD8+ T cells was related to CD8+ expression levels and cell size, i. e. IL-2-expressing cells were generally smaller with more intense CD8+ staining as compared with IFN-gamma-producing T cells. These data indicate that activated T lymphocytes are strongly committed in vivo to produce IFN-gamma or IL-2 and emphasizes the independent regulation of the two cytokine genes.

Anti-tumor immunity induced by murine melanoma cells transduced with the Mycobacterium tuberculosis gene encoding the 38-kDa antigen.

The Mycobacterium tuberculosis Ag38 gene, which encodes a highly immunogenic protein, was cloned into a retroviral vector in-frame with the leader and the transmembrane portion of the nerve growth factor receptor, and transduced into murine melanoma cell line B16-B78. Significant protection was observed in mice immunized with the transduced melanoma cells and subcutaneously challenged with parental melanoma cells since only 20% of mice developed tumors. Necroscopy of mice immunized with the transduced melanoma cells revealed dramatic inhibition of experimental metastases induced by intravenous (i.v.) inoculation of parental melanoma cells. Moreover, vaccination with transduced cells significantly prolonged survival of mice challenged i.v. with parental melanoma cells. These data indicate that the presence of the mycobacterial 38-kDa protein greatly enhances immunological recognition of structures expressed by the parental melanoma cells. Comparison of Th1 and Th2 responses in mice immunized with parental melanoma cells versus mice receiving the transduced cells revealed a clear predominance of Th1 responses when the Ag38 protein was endogenously expressed. This transduction approach may represent a promising immunotherapeutic strategy for the treatment of cancer patients.