Vaccination protocols designed to elicit anti-cancer immune responses have, in most cases, failed in producing tumor eradication and in prolonging patient survival. Usually in cancer vaccination, epitopes from one organism are included in the genome or linked with some protein of another (named carrier) in the hope that the immunogenic properties of the latter will boost an immune response to the former. However, recent results have demonstrated that injections of two different vectors encoding the same recombinant antigen generate high levels of specific immunity. Systematic comparison of the efficacy of different vaccination protocols has been hampered by technical limitations and a clear evidence that the use of multiple vectors has advantage over single carrier injections is lacking. We used a computational model to investigate the dynamics of the immune response to different anti-cancer vaccines based on randomly generated antigen/carrier compounds. More than 3000 simulations of the immune response to tumor were performed. Notably, the model has been extensively validated and it reproduces a relevant number of experimental observations. The model shows that when priming and boosting with the same construct, competition rather than cooperation develops amongst T cell clones of different specificities. Moreover, from the simulations, it appears that the sequential use of multiple carriers may generate more robust anti-tumor immune responses and may lead to effective tumor eradication in a higher percentage of cases. Our results provide a rational background for the design of novel strategies for the achievement of immune control of cancer.

Hairy cell leukemia (HCL) is a chronic B cell malignancy characterized by the diffuse infiltration of bone marrow and spleen by cells displaying a typical "hairy" morphology. However, the nature of the HCL phenotype and its relationship to normal B cells and to other lymphoma subtypes remains unclear. Using gene expression profiling, we show here that HCL displays a homogeneous pattern of gene expression, which is clearly distinct from that of other B cell non-Hodgkin lymphomas. Comparison with the gene expression profiles of purified normal B cell subpopulations, including germinal center (GC), pre-GC (naive), and post-GC (memory) B cells, shows that HCL cells are more related to memory cells, suggesting a derivation from this B cell population. Notably, when compared with memory cells, HCL cells displayed a remarkable conservation in proliferation, apoptosis, and DNA metabolism programs, whereas they appeared significantly altered in the expression of genes controlling cell adhesion and response to chemokines. Finally, these analyses have identified several genes that are specifically expressed in HCL and whose expression was confirmed at the protein level by immunocytochemical analysis of primary HCL cases. These results have biological implications relevant to the pathogenesis of this malignancy as well as clinical implications for its diagnosis and therapy.

Anaplastic large-cell lymphoma (ALCL), expressing anaplastic lymphoma kinase (ALK) as either a NPM-ALK fusion protein or an ALK-containing fusion protein, is a rare disease with distinctive molecular, pathological, and clinical features. 1 Shiota M Nakamura S Ichinohasama R et al. Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity. Blood. 1995; 86: 1954-1960 Crossref PubMed Google Scholar , 2 Falini B Pileri S Zinzani PL et al. ALK+ lymphoma: clinicopathological findings and outcome. Blood. 1999; 93: 2697-2706 PubMed Google Scholar , 3 Falini B Pulford K Pucciarini A et al. Lymphomas expressing ALK fusion protein(s) other than NPM-ALK. Blood. 1999; 94: 3509-3515 PubMed Google Scholar , 4 Stein H Foss HD Durkop H et al. CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood. 2000; 96: 3681-3695 PubMed Google Scholar , 5 Falini B Anaplastic large cell lymphoma: pathological, molecular and clinical features. Br J Haematol. 2001; 114: 741-760 Crossref PubMed Scopus (133) Google Scholar ALCL usually occurs during the first three decades of life, and presents as a disseminated (stages III–IV) disease with systemic symptoms. The disease is often confused with other lymphoma subtypes or even misdiagnosed as a reactive condition. 1 Shiota M Nakamura S Ichinohasama R et al. Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity. Blood. 1995; 86: 1954-1960 Crossref PubMed Google Scholar , 2 Falini B Pileri S Zinzani PL et al. ALK+ lymphoma: clinicopathological findings and outcome. Blood. 1999; 93: 2697-2706 PubMed Google Scholar , 3 Falini B Pulford K Pucciarini A et al. Lymphomas expressing ALK fusion protein(s) other than NPM-ALK. Blood. 1999; 94: 3509-3515 PubMed Google Scholar , 4 Stein H Foss HD Durkop H et al. CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood. 2000; 96: 3681-3695 PubMed Google Scholar , 5 Falini B Anaplastic large cell lymphoma: pathological, molecular and clinical features. Br J Haematol. 2001; 114: 741-760 Crossref PubMed Scopus (133) Google Scholar Many patients are cured by chemotherapy alone; chemoresistance is associated with poor prognosis. 1 Shiota M Nakamura S Ichinohasama R et al. Anaplastic large cell lymphomas expressing the novel chimeric protein p80NPM/ALK: a distinct clinicopathologic entity. Blood. 1995; 86: 1954-1960 Crossref PubMed Google Scholar , 2 Falini B Pileri S Zinzani PL et al. ALK+ lymphoma: clinicopathological findings and outcome. Blood. 1999; 93: 2697-2706 PubMed Google Scholar , 3 Falini B Pulford K Pucciarini A et al. Lymphomas expressing ALK fusion protein(s) other than NPM-ALK. Blood. 1999; 94: 3509-3515 PubMed Google Scholar , 4 Stein H Foss HD Durkop H et al. CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features. Blood. 2000; 96: 3681-3695 PubMed Google Scholar , 5 Falini B Anaplastic large cell lymphoma: pathological, molecular and clinical features. Br J Haematol. 2001; 114: 741-760 Crossref PubMed Scopus (133) Google Scholar Here, we report the clinical and pathological characteristics of a patient with chemoresistant ALK+ ALCL, and its successful treatment with haploidentical peripheral-blood stem-cell (PBSC) transplantation.

Patients undergoing full haplotype-mismatched hematopoietic transplantations may experience severe intractable invasive fungal infections. To verify whether an imbalanced production of T-helper 1 (TH1) and TH2 cytokines may be responsible for susceptibility to fungal infections, C3H/HeJ (H-2(k)) recipient mice were lethally irradiated, received transplantations with T-cell-depleted allogeneic bone marrow (BM) cells from mice of H-2(d) haplotype, and were infected with Candida albicans. At different time-points after transplantation, mice were assessed for pattern of TH cytokine production and susceptibility to infection. The results show that a long-term, donor-type chimerism was achieved as early as 2 weeks after BM transplantation (BMT), at the time when high-level production of TH2 cytokines (interleukin-4 [IL-4] and IL-10) and impaired production of TH1 cytokines (interferon-gamma [IFN-gamma] and IL-12] were observed. At this time, mice were highly susceptible to both disseminated and mucosal infections, as indicated by decreased survival, uncontrolled fungal growth, and failure to develop protective TH1 immunity. However, a predominant production of TH1 cytokines was observed by week 5 after BMT, at the time when mice developed donor-type protective TH1 responses and were resistant to infections. Therapeutic ablation of IL-4 or IL-10 greatly increased resistance to candidiasis. These results indicate that a dysregulated production of TH cytokines occurs in mice undergoing T-cell-depleted allogeneic BMT. The transient predominant production of TH2 cytokines over that of IL-12 impaired the ability of mice to develop antifungal TH1 resistance, an activity that could be efficiently restored upon treatment with TH2 cytokine antagonists.

We constructed a functional MoMuLV-based bicistronic retroviral vector encoding the herpes simplex virus type I thymidine kinase gene, which induces sensitivity to the prodrug ganciclovir (gcv), and the reporter beta -galactosidase gene (MFG-tk-IRES-lacZ). The U937 histiocytic cell line was transduced with this vector, and a clone (VB71) with high-level transgene expression was selected, Severe combined immunodeficient (SCID) mice were injected with VB71 cells to evaluate the role of long terminal repeat methylation in transgene silencing in vivo and to see whether 5-azacytidine (5' aza-C) demethylating agent prevented it.We found 5' aza-C maintained gene expression at high level in vitro. In vivo, time to tumor onset was significantly longer in SCID mice receiving the VB71 cells, 5' aza-C, and gcv compared with animals treated with either 5' aza-C or gcv alone. The number of injected turner cells influences tumor onset time and the efficacy of 5' aza-C and gcv treatment. The standard gcv treatment schedule (10 mg/kg from d + 1 until the onset of tumor) controlled tumor onset better than short-term treatment with high doses. In conclusion, the results extend our previous findings that transgene methylation in vivo may be prevented viith an appropriate schedule of 5' aza-C and gcv.

Retroviral sequence can silence transgene expression in vitro and in vivo. We report that this effect can be efficiently prevented by in vivo administration of the demethylating agent 5-azacytidine (aza-C). We engineered the U937 human cell line with a retroviral vector consisting of the thymidine kinase suicide gene (tk), which induces sensitivity to ganciclovir (gcv) and through an IRES sequence, the bacterial beta-galactosidase gene (lacZ) as a marker gene. About 90% of the U937 cells expressed the transgene. By injecting the transduced U937 cells in severe combined immunodeficient disease (SCID) mice, we generated a tumour which, during in vivo treatment with aza- C, maintained the high expression of lacZ and tk genes at the baseline values. LacZ-positive cells in the tumour masses after death was weak (1–2%) in the control group, while in mice treated with aza-C it was maintained at 90%. The delay in tumour onset was significanly longer when animals were treated with both aza-C and gcv (P < 0.0001) compared with animals treated with gcv or with aza-c alone. the prevention of silencing phenomena has important implications for gene therapy, because an efficient transduction associated with appropriate drug therapy, might be a powerful strategy for successful application of gene therapy protocols.