[Photocarcinogenesis-molecular mechanisms and practical relevance]. / Photokarzinogenese ­ Molekulare Mechanismen und praktische Relevanz.

Ultraviolet (UV) radiation is the main risk factor for the development of melanocytic and nonmelanocytic skin cancer. UVA and UVB radiation are of particular importance in photocarcinogenesis. Depending on the wavelength, mechanisms of tumor initiation and promotion include direct DNA damage and proinflammatory processes. In recent years, the number of skin cancer cases in Germany has continuously increased. In addition to regular skin check-ups, use of suitable textile protection and sunscreens play a central role in the prevention of cancer development. As dermatologists, it is our task to regularly inform our patients about the consequences of excessive sun exposure and to adequately inform them about necessary protective devices.

Acral lentiginous melanoma: a skin cancer with unfavourable prognostic features. A study of the German central malignant melanoma registry (CMMR) in 2050 patients.

BACKGROUND: Acral lentiginous melanoma (ALM) is one of the four major subtypes in cutaneous melanoma (CM). Although ALM has a poorer prognosis than other CM subtypes, the prognostic factors associated with ALM have only been verified in small-sized cohorts because of the low incidence of ALM worldwide. OBJECTIVES: To investigate the clinical characteristics of ALM and to evaluate their prognostic values based on a large dataset from the Central Malignant Melanoma Registry (CMMR) of the German Dermatologic Society. METHODS: The Kaplan-Meier method was used to estimate the potential influence of clinical and histological parameters on ALM disease-specific survival (DSS) curves, which were compared using the log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors for DSS. RESULTS: In total, 2050 patients with ALM were identified from 58 949 patients with CM recorded by the CMMR with follow-up data. In multivariate analyses, age (P = 0·006), ulceration (P = 0·013), tumour thickness (P < 0·001) and tumour spread (P < 0·001) turned out to be significant prognostic factors for DSS in ALM whereas sex, nevus association and level of invasion were not independent factors. CONCLUSIONS: ALM has the same prognostic factors as other subtypes of melanoma. Unfavourable prognosis probably derives from the delay in diagnosis in comparison with other melanoma subtypes.

Anti-inflammatory activity of topical THC in DNFB-mediated mouse allergic contact dermatitis independent of CB1 and CB2 receptors.

BACKGROUND: ∆(9) -Tetrahydrocannabinol (THC), the active constituent of Cannabis sativa, exerts its biological effects in part through the G-protein-coupled CB1 and CB2 receptors, which were initially discovered in brain and spleen tissue, respectively. However, THC also has CB1/2 receptor-independent effects. Because of its immune-inhibitory potential, THC and related cannabinoids are being considered for the treatment of inflammatory skin diseases. Here we investigated the mechanism of the anti-inflammatory activity of THC and the role of CB1 and CB2 receptors. METHODS: We evaluated the impact of topically applied THC on DNFB-mediated allergic contact dermatitis in wild-type and CB1/2 receptor-deficient mice. We performed immunohistochemical analyses for infiltrating immune cells and studied the influence of THC on the interaction between T cells, keratinocytes and myeloid immune cells in vitro. RESULTS: Topical THC application effectively decreased contact allergic ear swelling and myeloid immune cell infiltration not only in wild-type but also in CB1/2 receptor-deficient mice. We found that THC (1) inhibited the production of IFNγ by T cells, (2) decreased the production of CCL2 and of IFNγ-induced CCL8 and CXL10 by epidermal keratinocytes and (3) thereby limited the recruitment of myeloid immune cells in vitro in a CB1/2 receptor-independent manner. CONCLUSIONS: Topically applied THC can effectively attenuate contact allergic inflammation by decreasing keratinocyte-derived pro-inflammatory mediators that orchestrate myeloid immune cell infiltration independent of CB1/2 receptors. This has important implications for the future development of strategies to harness cannabinoids for the treatment of inflammatory skin diseases.

Efficacy of low-dose methotrexate in the treatment of dermatomyositis skin lesions.

A limited number of case series has indicated that methotrexate (MTX) might be a useful drug in the treatment of dermatomyositis (DM), a rare autoimmune disease involving the skin and muscles. However, these earlier studies mainly focused on the efficacy of MTX on DM muscular symptoms. To analyse the efficacy of MTX on skin lesions in DM, the records of 34 patients with DM seen between 2004 and 2009 were retrospectively analysed, and the DM skin disease activity at different time points was determined, with specific focus on cutaneous features using the validated Cutaneous Dermatomyositis Activity Index (CDASI) score. The lesional inflammation was scored in primary skin biopsies. Additionally, we performed a systematic review of the available literature. In our series, 11 patients with DM received MTX, and in 8 of them, MTX led to a significant reduction of the DM skin lesions. CDASI scores decreased from 15.7 to 6.4 (P < 0.01) within 2-3 months, supporting the effectiveness of MTX in skin disease in DM. The lymphocytic infiltrate in primary skin lesions of MTX responders was significantly more pronounced than that in nonresponders. These results indicate that MTX might be an effective drug to treat the cutaneous symptoms of DM, as measured by the validated CDASI. Interestingly, MTX responders histologically presented a significantly stronger lesional lymphocytic inflammatory infiltrate than did nonresponders. These findings suggest that the functional inhibition of lymphocyte migration in the skin might be an important mechanism of MTX in the treatment of DM.

Interferon-α stimulates TRAIL expression in human keratinocytes and peripheral blood mononuclear cells: implications for the pathogenesis of cutaneous lupus erythematosus.

BACKGROUND: The tumour necrosis factor-related apoptosis-inducing ligand TRAIL has been shown to participate in the pathogenesis of systemic lupus erythematosus (SLE). The accumulation of apoptotic cell debris has been hypothesized to induce this autoimmune inflammation, and TRAIL may trigger this programmed cell death. Furthermore, TRAIL is among the interferon (IFN)-regulated genes which are typically expressed in the peripheral blood of patients with acute SLE. OBJECTIVES: As an inappropriate activation of the type I IFN system plays an important role in both SLE and cutaneous lupus erythematosus (CLE) subsets, we hypothesized that TRAIL might also participate in the pathogenesis of CLE. METHODS: Immunohistochemistry and immunofluorescence analyses were used to identify and localize TRAIL-expressing cells in CLE skin specimens. TRAIL expression in peripheral blood mononuclear cells (PBMC) isolated from patients with CLE was measured by flow cytometry. The impact of IFN-α treatment on TRAIL expression by keratinocytes and PBMC was evaluated by reverse transcription-polymerase chain reaction and flow cytometry. RESULTS: Keratinocytes are beside CD11c+ and BDCA2+ dendritic cells the major TRAIL-expressing cells in CLE lesions. TRAIL is upregulated on the surface of circulating CD11c+ PBMC isolated from patients with CLE. Treatment of keratinocytes and PBMC with recombinant IFN-α strongly enhances TRAIL expression by these cells. The proapoptotic TRAIL receptor R1 is expressed by keratinocytes in CLE skin lesions. CONCLUSIONS: TRAIL is strongly expressed in the skin and the blood of patients with CLE and may trigger the apoptotic death of kerationcytes in CLE via the TRAIL receptor R1. An IFN-α-induced TRAIL expression may in this way participate in the pathogenesis of CLE.

Enhanced CCR5+/CCR3+ T helper cell ratio in patients with active cutaneous lupus erythematosus.

Cutaneous lupus erythematosus (CLE) is characterized by enhanced interferon α (IFNα) levels in serum and in tissue. Since IFNα promotes a Th1-biased immune response, we hypothesized that a Th1-associated chemokine receptor profile should be a typical finding in patients with active CLE. Therefore, peripheral blood mononuclear cells were isolated from patients with different CLE subsets (n = 15), healthy controls (n = 13) and patients under immunotherapy with IFNα (n = 7). T helper cells were analysed by flow cytometry for the expression of the chemokines receptor CCR5, indicative for Th1 cells, and of CCR3, indicating Th2. In addition, intracellular levels of the type I IFN-inducible MxA protein were measured. Patients with widespread active CLE skin lesions had a significantly increased expression of CCR5, whereas expression of CCR3 was decreased when compared with healthy controls. MxA expression was significantly enhanced in all investigated CLE subtypes, with the highest levels in patients with widespread skin lesions. The enhanced CCR5/CCR3 ratio closely correlated with the MxA levels in peripheral lymphocytes and with disease activity. Our analyses revealed that active CLE is associated with a systemic type I IFN effect that appears to induce a shift towards a Th1-associated chemokine receptor profile. The CCR5/CCR3 T-helper cell ratio might therefore represent an indirect marker for the disease activity in CLE.

Protective role of palmitoylethanolamide in contact allergic dermatitis.

BACKGROUND: Palmitoylethanolamide (PEA) is an anti-inflammatory mediator that enhances the activation by anandamide (AEA) of cannabinoid receptors and transient receptor potential vanilloid type-1 (TRPV1) channels, and directly activates peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In mice, 2,4-dinitrofluorobenzene (DNFB)-induced contact allergic dermatitis (CAD) in inflamed ears is partly mediated by the chemokine Monocyte Chemotactic Protein-2 (MCP-2) and accompanied by elevation of AEA levels. No datum is available on PEA regulation and role in CAD. OBJECTIVE: We examined whether PEA is produced during DNFB-induced CAD, and if it has any direct protective action in keratinocytes in vitro. METHODS: Eight- to ten-week-old female C57BL/6J wild-type and CB(1)/CB(2) double knock-out mice were used to measure PEA levels and the expression of TRPV1, PPAR-alpha receptors and enzymes responsible for PEA biosynthesis and degradation. Human keratinocytes (HaCaT) cells were stimulated with polyinosinic polycytidylic acid [poly-(I:C)], and the expression and release of MCP-2 were measured in the presence of PEA and antagonists of its proposed receptors. RESULTS: 2,4-Dinitrofluorobenzene increased ear skin PEA levels and up-regulated TRPV1, PPAR-alpha and a PEA-biosynthesizing enzyme in ear keratinocytes. In HaCaT cells, stimulation with poly-(I:C) elevated the levels of both PEA and AEA, and exogenous PEA (10 microM) inhibited poly-(I:C)-induced expression and release of MCP-2 in a way reversed by antagonism at TRPV1, but not PPAR-alpha. PEA (5-10 mg/kg, intraperitoneal) also inhibited DNFB-induced ear inflammation in mice in vivo, in a way attenuated by TRPV1 antagonism. CONCLUSIONS: We suggest that PEA is an endogenous protective agent against DNFB-induced keratinocyte inflammation and could be considered for therapeutic use against CAD.

Pathogenesis of cutaneous lupus erythematosus: common and different features in distinct subsets.

The term 'cutaneous lupus erythematosus' (CLE) comprises several related autoimmune skin disorders, defined as 'specific' skin manifestations of lupus erythematosus (LE). The spectrum of clinical presentation of CLE is wide, reaching from mild erythema to disseminated scarring skin lesions. There is increasing knowledge concerning the pathogenesis of LE skin lesions and it has been shown that a complex network of cutaneous cytokines, chemokines and adhesion molecules orchestrate and promote tissue injury observed in LE skin lesions. However, a complete understanding of the diverse pathophysiological mechanisms in the different CLE subsets does not exist. Here we review the main pathological features described in CLE patients against the background of the clinical diversity of different CLE subtypes.

[Multiple skin cancers in a patient treated with hydroxyurea]. / Multiple kutane Neoplasien nach Therapie mit Hydroxyurea.

A 62-year-old patient treated for 9 years with hydroxyurea for chronic myeloproliferative disease developed multiple cutaneous neoplasms. Hydroxyurea minimizes DNA synthesis via inhibition of the enzyme ribonucleotide reductase and is used to treat hematological malignancies. The most important and severe side-effect is a dose-dependent myelodepression. An association with multiple skin tumors has been reported. The presented case emphasizes this potential risk of hydroxyurea therapy. Continuous dermatologic monitoring of patients treated with hydroxyurea is recommended.

Deletion of ADAM-9 in HGF/CDK4 mice impairs melanoma development and metastasis.

ADAM-9 is a metalloproteinase expressed in peritumoral areas by invading melanoma cells and by adjacent peritumoral stromal cells; however, its function in stromal and melanoma cells is not fully understood. To address this question in vivo in a spontaneous melanoma model, we deleted ADAM-9 in mice carrying the hepatocyte growth factor (Hgf) transgene and knock-in mutation Cdk4R24C/R24C, demonstrated to spontaneously develop melanoma. Spontaneous melanoma arose less frequently in ADAM-9-deleted mice than in controls. Similarly reduced tumor numbers (although with faster growth kinetics) were detected upon induction of melanoma with 7,12-dimethylbenz[a]anthracene (DMBA). However, more lesions were induced at early time points in the absence of ADAM-9. Increased initial and decreased late tumor numbers were paralleled by altered tumor cell proliferation, but not apoptosis or inflammation. Importantly, significantly reduced lung metastases were detected upon ADAM-9 deletion. Using in vitro assays to address this effect mechanistically, we detected reduced adhesion and transmigration of ADAM-9-silenced melanoma cells to/through the endothelium. This implies that ADAM-9 functionally and cell autonomously mediates extravasation of melanoma cells. In vitro and in vivo we demonstrated that the basement membrane (BM) component laminin ß3-chain is a direct substrate of ADAM-9, thus contributing to destabilization and disruption of the BM barrier during invasion. In in vitro invasion assays using human melanoma cells and skin equivalents, depletion of ADAM-9 resulted in decreased invasion of the BM, which remained almost completely intact, as shown by continuous staining for laminin ß3-chain. Importantly, supplying soluble ADAM-9 to the system reversed this effect. Taken together, our data show that melanoma derived ADAM-9 autonomously contributes to melanoma progression by modulating cell adhesion to the endothelium and altering BM integrity by proteolytically processing the laminin-ß3 chain. This newly described process and ADAM-9 itself may represent potential targets for anti-tumor therapies.

Comparison of recombinant adenovirus and synthetic peptide for DC-based melanoma vaccination.

Optimal strategies for antigen-specific melanoma vaccination are currently being defined in experimental mouse models. Using a single H2-K(b)-binding peptide epitope derived from the melanosomal enzyme tyrosinase-related protein 2 (TRP2) in C57BL/6 mice, we show that adenovirus-transduced dendritic cells (DC) are clearly superior to peptide-pulsed DC for the induction of CD8+ T cells and antimelanoma immunity. Vaccine efficacy strictly depended on the presence of linked CD4+ T-cell help during the priming but not the effector phase of the immune response. These results provide important information for the translation of melanoma vaccine strategy in future clinical applications.

Depletion of CD25(+) CD4(+) T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8(+) T-cell-dependent immune defense of B16 melanoma.

Transduction of B16 melanoma cells with IFN alpha (B16-IFN alpha) enhances CD8(+) T-cell-dependent tumor immunity in mice, resulting in delayed outgrowth in vivo. Here we provide evidence that CD4(+) T cells down-regulate the IFN alpha-induced tumor immune defense. Importantly, depletion of regulatory CD25(+) CD4(+) T cells prevented growth of B16-IFN alpha in most mice and promoted long-lasting protective tumor immunity. Rejection of B16-IFN alpha could also be achieved with therapeutic injections of dendritic cells genetically engineered to express the melanoma antigen tyrosinase-related protein 2. These results support the development of novel strategies for the immunotherapy of melanoma using IFN alpha in combination with elimination of regulatory T cells or antigen-specific immunization.

Gene-based strategies for the immunotherapy of cancer.

T lymphocytes play a crucial role in the host's immune response to cancer. Although there is ample evidence for the presence of tumor-associated antigens on a variety of tumors, they are seemingly unable to elicit an adequate antitumor immune response. Modern cancer immunotherapies are therefore designed to induce or enhance T cell reactivity against tumor antigens. Vaccines consisting of tumor cells transduced with cytokine genes in order to enhance their immunogenicity have been intensely investigated in the past decade and are currently being tested in clinical trials. With the development of novel gene transfer technologies it has now become possible to transfer cytokine genes directly into tumors in vivo. The identification of genes encoding tumor-associated antigens and their peptide products which are recognized by cytotoxic T lymphocytes in the context of major histocompatibility complex class I molecules has allowed development of DNA-based vaccines against defined tumor antigens. Recombinant viral vectors expressing model tumor antigens have shown promising results in experimental models. This has led to clinical trials with replication-defective adenoviruses encoding melanoma-associated antigens for the treatment of patients with melanoma. An attractive alternative concept is the use of plasmid DNA, which can elicit both humoral and cellular immune responses following injection into muscle or skin. New insights into the molecular biology of antigen processing and presentation have revealed the importance of dendritic cells for the induction of primary antigen-specific T cell responses. Considerable clinical interest has arisen to employ dendritic cells as a vehicle to induce tumor antigen-specific immunity. Advances in culture techniques have allowed the generation of large numbers of immunostimulatory dendritic cells in vitro from precursor populations derived from blood or bone marrow. Experimental immunotherapies which now transfer genes encoding tumor-associated antigens or cytokines directly into professional antigen-presenting cells such as dendritic cells are under evaluation in pre-clinical studies at many centers. Gene therapy strategies, such as in vivo cytokine gene transfer directly into tumors as well as the introduction of genes encoding tumor-associated antigens into antigen-presenting cells hold considerable promise for the treatment of patients with cancer.

DNA immunization targeting the skin: molecular control of adaptive immunity.

DNA-based immunization represents a novel approach for vaccine development. Recombinant DNA techniques are used to clone DNA sequences encoding antigens of choice into eukaryotic expression plasmids, which are readily and economically amplified in bacteria and recovered with a high degree of purity. For immunization, plasmid DNA is either coated onto microscopic gold particles and bombarded into skin using a gene gun or injected into skin or muscle. Expression of administered genes results in the induction of humoral and cellular immune responses against the encoded antigen. DNA immunization is capable of inducing protective immunity in a number of animal models of infectious disease and cancer. Recent studies suggest that antigen-specific cytotoxic T lymphocyte induction occurs through the presentation of appropriate peptides in the context of major histocompatibility complex molecules on bone marrow-derived professional antigen presenting cells. Following DNA inoculation into the skin, Langerhans cells and/or dermal dendritic cells are believed to acquire the newly synthesized antigen, either through direct transfection or via antigen uptake from transfected keratinocytes, and migrate to regional lymph nodes where they stimulate primary T cell responses. The nature of the immune response depends on the route, method, and timing of DNA delivery and can also be influenced by co-delivery of plasmids encoding immunomodulating cytokines like IFN-alpha, IL-2, or IL-12 and costimulatory molecules like B7-1. While many aspects of the biology of cutaneous DNA immunization remain unknown, the skin appears to offer unique potential as a target for DNA-based immunization.

Induction of tumor antigen-specific immunity using plasmid DNA immunization in mice.

We have evaluated the ability of bioballistic "gene gun" immunization of mice with plasmid DNA encoding clinically relevant tumor antigens to induce protective antitumor immunity. Mice immunized with plasmid cDNA encoding the cervical carcinoma-associated human papillomavirus 16-E7 gene product exhibited potent anti-E7-specific cytotoxic T lymphocytes and were protected completely against a subsequent challenge with the E7+ C3 sarcoma. Of perhaps greater clinical interest, genetic immunization using cDNA encoding the normal, germline-encoded murine melanosomal protein tyrosinase-related protein-2 (TRP-2) resulted in delayed outgrowth of TRP-2+ B16 melanoma in mice and was associated with an in vivo activation of TRP-2-specific cytotoxic T lymphocytes. Codelivery of plasmid cDNA encoding TRP-2 and the T helper 1-biasing cytokine murine interleukin-12 considerably enhanced the antitumor efficacy of these gene-based melanoma vaccines.

Induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12.

Direct intratumoral (i.t.) injection of adenoviruses (Ads) expressing specific immunostimulatory cytokines represents an attractive strategy for the clinical implementation of cytokine gene therapy of cancer. Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells and promotes a T helper 1-like immune response. We have constructed an Ad vector (AdCMV-mIL-12) containing both chains of the murine IL-12 (mIL-12) gene linked by an internal ribosomal entry site sequence under the transcriptional control of the cytomegalovirus immediate-early gene promoter, which is able to mediate the transient expression of very high levels of biologically active mIL-12 both in vitro and in vivo. An i.t. injection of 4x10(8) plaque-forming units of AdCMV-mIL-12 resulted in a complete regression of day 7 established subcutaneous MC38 murine adenocarcinomas and MCA205 murine fibrosarcomas. Treated animals rejected a subsequent rechallenge with MC38 and MCA205, respectively, demonstrating the induction of long-lasting antitumor immunity. Specific antitumor cytotoxic T lymphocyte reactivity was detected in splenocytes harvested from treated animals. A significant increase in the numbers of both CD4+ and CD8+ T cells in the AdCMV-mIL-12-infected tumors was observed. Ad-mediated IL-12 gene therapy was also associated with measurable serum levels of mIL-12 and profound changes in the composition of splenic lymphocytes. Taken together, these results demonstrate the feasibility and efficacy of delivering IL-12 directly i.t. using a recombinant adenoviral vector.

The immunology of cutaneous DNA immunization.

The expression of a foreign protein in the skin or muscle following direct in vivo gene transfer results in the induction of potent cellular and humoral immune responses and represents a novel approach for vaccine development. This strategy, now known as genetic or DNA immunization, can be performed either by the bombardment of skin with plasmid DNA coated onto microscopic gold particles using a gene gun, or by injection of muscle with plasmid DNA in saline solution. DNA immunization leads to protective immunity in a variety of rodent models of infectious disease and cancer. Here we will review recent advances in this rapidly expanding research area. Most importantly, the cellular and molecular events leading to the induction of immune responses following DNA inoculation into the skin are now understood in much greater detail. It has become clear that the dendritic cell (DC) system of bone marrow-derived antigen presenting cells (APC) is the initiator of cellular immune responses. Evidence has accumulated that cutaneous DCs are directly transfected leading to endogenous antigen synthesis and processing. The gene transfer procedure results in the activation of DCs and initiates migration to regional lymph nodes, where antigen-expressing DCs efficiently stimulate proliferation of antigen-specific CD8+ as well as CD4+ T-lymphocytes. The nature of the immune response following plasmid DNA immunization may be manipulated by co-delivery of plasmids encoding immunomodulatory cytokines like (interferon) IFNalpha, IL-2 or IL-12 and costimulatory molecules like B7-1. Molecular re-engineering of antigen-encoding plasmids allows for specific targeting of antigen expression into the antigen processing machinery of DC for optimal presentation to CD8+ and CD4+ T-lymphocytes. DNA immunization is currently being developed for the prevention and treatment of a number of infectious diseases and cancer.

Induction of dendritic cell maturation and modulation of dendritic cell-induced immune responses by prostaglandins.

Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system. In this study we investigated the effects of various prostaglandins (PG) on the stimulatory capacity of DC. DC were generated from peripheral progenitor cells in the presence of IL-4 and GM-CSF and stimulated with IL-1, IL-6 and TNF-alpha on day 7. Simultaneously, PG (PGD(2), PGE(1), PGE(2), PGF(2 alpha), PGI(2)) were added at various concentrations (10(-5) to 10(-9) M) on day 7. In all experiments, PGE(2) had the most potent influence on the maturation of the DC, followed by other PG in the order PGE(1) > PGD(2) > PGF(2 alpha) > PGI(2). In addition, the expression of the surface molecules CD40, CD54, CD58, CD80, CD83, CD86 and the MHC class II molecules was upregulated after stimulation with PG. Analysis of DC supernatants after treatment with PG demonstrated significantly higher amounts of the proinflammatory cytokines IL-1 beta, IL-6, TNF-alpha, and IL-12. Addition of PG to DC induced a markedly enhanced proliferation of both naive and activated CD4(+) and CD8(+) T cells in alloantigen-induced MLR assays. Assessment of coculture supernatants after restimulation revealed significantly higher amounts of the Th1-cytokines IL-2 and IFN-gamma and only minimal amounts of IL-4 compared to control cells. No production of IL-10 was observed. The effects of PG on the maturation of DC and enhanced T-cell proliferation could be mimicked by db-cAMP and forskolin, indicating that they were due to elevated cAMP levels. Collectively, our data show that members of the PG family promote the differentiation of DC and enhance their capacity to induce a Th1 immune response.

Particle-Mediated Gene Transfer into Dendritic Cells : A Novel Strategy for the Induction of Immune Responses against Tumor Antigens.

The expression of a foreign protein in the skin following direct in vivo gene transfer results in the induction of potent cellular and humoral immune responses. This strategy, now known as genetic or DNA immunization, was first described by Johnston et al. in 1992. They reported that bombardment of murine skin with an expression plasmid encoding human growth hormone coated onto microscopic gold particles using a gene gun resulted not only in the systemic delivery of the molecule, but also in the induction of antigenspecific antibody responses (1). It is now well established that DNA immunization by particle-mediated gene transfer promotes broad-based and long-lasting antigen-specific immune responses capable of protecting against challenges with infectious agents and tumor cells in rodents [reviewed in (2,3)]. Importantly, gene gun immunization elicits both humoral and cellular immunity, consisting of antibody responses specific for conformational determinants, as well as, antigen-specific CD8(+)cytotoxic T cells and CD4(+)T-helper cells. For this reason, it represents an attractive novel approach for the clinical development of prophylactic and therapeutic vaccines against certain infectious diseases and tumors. We are currently investigating, like many other tumor immunologists, whether DNA-based immunization can lead to the induction of potent cellular and humoral immune responses against defined tumor antigens that are associated with tumor rejection activity in vivo.