Adenovirus-mediated ex vivo immunogene and in vivo combination gene therapy strategies induce a systemic anti-tumor immune defense in the mouse B16 melanoma model.
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Adenoviridae↗
Biomedical subjects
B Bonnekoh
Publications and source records attributed to B Bonnekoh.
Ex vivo and in vivo adenovirus-mediated gene therapy strategies induce a systemic anti-tumor immune defence in the B16 melanoma model.
The efficacy of adenovirus-mediated gene therapy for treatment of metastatic B16 melanomas, established in syngeneic C57BL/6 mice, was assessed via an ex vivo cytokine vaccine approach or via an in vivo strategy utilizing combination cytokine/herpes simplex virus-thymidine kinase (HSV-tk) suicide gene delivery and treatment with ganciclovir (GCV). In the ex vivo tumor vaccine approach, B16 melanoma cells, transduced in vitro by adenovirus containing either interleukin (IL)-2, granulocyte-macrophage colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha cytokine genes and gamma irradiated, were subcutaneously injected into the flank and a distant subcutaneous challenge injection of unmodified B16 melanoma cells was performed 15 d later. Significant reductions in challenge tumor volume were observed in the IL-2 group (75% reduction; p = 0.02) and in the GM-CSF group (88% reduction; p = 0.0006), whereas the effect for tumor necrosis factor-alpha was not statistically significant. In the in vivo treatment of established melanomas, this cytokine approach was combined with a suicide gene therapy and subcutaneous B16 melanomas were directly injected with (i) IL-2/recombinant, replication-deficient adenovirus (adv) and thymidine kinase (tk)/adv, (ii) GM-CSF/adv, IL-2/adv, and tk/adv, or (iii) control beta-galactosidase (beta-gal)/adv and tk/adv. After intraperitoneal application of GCV (10 mg per kg) for 6 d, the residual tumor masses were excised and the animals challenged with unmodified B16 cells. Challenge tumor growth was reduced by 56% for the IL-2/tk/adv/GCV treatment (p = 0.041) and by 77% for the GM-CSF/IL-2/tk/adv/GCV treatment p (p = 0.037), in comparison with the beta-gal/tk/GCV control group. These data may hold significant promise for the development of effective ex vivo and in vivo gene therapy modalities to counter the highly metastatic nature of human melanoma.
Adenoviridae↗
Immunological gene therapy approaches for malignant melanoma. 1. Tumor-immunological background.
Gene therapy approaches pursuing immunological strategies for the treatment of malignant melanoma play major roles in the current efforts to explore the potential benefits of gene transfer technologies for medicine. This may be explained by the nearly complete resistance of advanced metastatic melanoma towards conventional non-surgical treatment modalities, and the particular immunogenicity of melanoma in connection with a presumed immuno-gene therapeutic 'field effect'. The latter relates to the potency of the immune system to amplify gene transfer effects that are limited due to the imperfection of the currently available gene delivery systems. The ongoing clinical trials focus predominantly on treatment safety and tolerability rather than efficacy. The corresponding tumor-immunological background is reviewed, focusing on a treatment concept centred on tumor-reactive, cytotoxic CD8+ T effector cells.
CD8-Positive T-Lymphocytes↗
Immunological gene therapy approaches for malignant melanoma. 2. Preclinical studies and clinical strategies.
Immuno-gene therapy approaches for the treatment of malignant melanoma are categorized into two major subgroups according to an active or passive immunological principle. Active immuno-gene therapy is subdivided into melanoma cell vaccines, DNA-based vaccinations and the treatment of pre-existing tumor tissue by cell-mediated or direct transfer of cytokine and/or cell surface signal genes. Passive immuno-gene therapy, employing an adoptive treatment with in vitro activated and expanded anti-tumor effector cells, involves two major application fields for gene transfer techniques, first the genetic modification of the effector cells, and second the in vivo amplification of pre-effector cells by procedures also used in active immuno-gene therapy. Corresponding preclinical studies are reviewed. The clinical studies inaugurated during the last few years are mostly still ongoing and focus on treatment safety and tolerability rather than efficacy. A recent trend is emerging to explore recombinant adenovirus and vaccinia virus vectors particularly with regard to in vivo gene transfer applications. Overall, immuno-gene therapy of melanoma is still in a highly experimental stage of development but may become a safe, efficacious and practical adjuvant treatment modality in the future.
Animals↗
Human keratinocyte culture from the peritonsillar mucosa.
Tonsillectomy tissue can be used as a routine source for cultures of oropharyngeal keratinocytes. In so doing, a peritonsillar strip of unaltered mucosa was dissected in the upper submucosa. Subsequent trypsinization yielded 7.0 +/- 3.4 x 10(6) keratinocytes per bilateral tonsillectomy. Keratinocyte attachment and growth in primary culture were promoted by sublethally irradiated 3T3 murine fibroblasts. Three subcultures could be performed without a feeder layer and were characterized by a population doubling time of 4.5 days during log growth phase. Electrophoretic and immunoblot analysis of the third subculture revealed a strong expression of keratin pairs 5/14 and 6/16 as well as keratins 7 and 19, whereas keratins 8/18 were expressed less intensely. The lowest intensity, was found for keratin 13, which is known to be indicative of the differentiated mucosa. The culture technique thus provides an easily available in vitro model for morphological and functional studies on the epithelial compartment of human oropharyngeal mucosa.
3T3 Cells↗
Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma.
To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.
Adenoviridae↗
Altered immunohistochemical expression of small proteoglycans in the tumor tissue and stroma of basal cell carcinoma.
Small proteoglycans have been shown to act as receptors for matrix molecules or growth factors and to influence the attachment and the migration of cells. We therefore report here on the immunocytochemical expression of three small proteoglycans, i.e., decorin, biglycan, and the recently described PG-100, in normal human skin and in basal cell carcinoma. In normal human skin, staining for decorin revealed expression throughout the dermis with an increased signal in the papillary dermis, whereas no expression was observed in the epidermis. Biglycan and PG-100 were mainly detected in the epidermis, with biglycan being expressed only in suprabasal layers. In addition, biglycan could be detected in a narrow zone below the basement membrane. In tissue specimens obtained from 12 basal cell carcinomas, the expression of biglycan and PG-100 was absent or strongly down-regulated in the tumor tissue. Tumor cells thus displayed a staining pattern similar to that found on the basal cells of normal human skin. In the stroma surrounding the tumor, however, the expression of biglycan and to a lesser degree decorin was increased when compared with normal human dermis. The increased deposition appears to be due to an increased synthesis of these molecules, as total RNA extracted from basal cell carcinoma tissue revealed an induction of biglycan and decorin mRNA. This study indicates that the expression of proteoglycans in basal cell carcinoma tumor cells and in tumor stroma is altered from that in normal skin.
Animals↗
Up-regulation of keratin 17 expression in human HaCaT keratinocytes by interferon-gamma.
The immortalized human keratinocyte cell line HaCaT was used to assess the effect of interferon-gamma (IFN-gamma) on expression of keratin K17. Both IFN-gamma and K17 have been implicated in the pathophysiology of psoriasis. Western and quantitative enzyme-linked immunosorbent assay analyses demonstrated increasing induction of K17 protein by 48 h exposure to IFN-gamma at concentrations of 10, 50, and 250 U/ml. At 50 U/ml IFN-gamma, immunohistochemical analysis revealed numerous K17-positive foci, whereas in situ hybridization demonstrated K17 message in the majority of cells. In addition, at low (5 U/ml) concentrations of IFN-gamma, cell proliferation and protein synthesis decreased, as determined by 3H-thymidine labeling and 14C-amino acid uptake. These data suggest that aberrant K17 expression observed in psoriatic lesions may be a consequence of IFN-gamma overexpression, and that the HaCaT cell line may be a useful in vitro model system to elucidate the underlying mechanisms.
Antigens, Differentiation↗
Inhibition of melanoma growth by adenoviral-mediated HSV thymidine kinase gene transfer in vivo.
To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.
Adenoviridae↗
Antiproliferative and cytotoxic profiles of antipsoriatic fumaric acid derivatives in keratinocyte cultures.
Oral administration with complex mixtures of fumaric acid derivatives is known to have antipsoriatic efficacy. The present studies aimed to clarify the mode of action and toxicity of the individual compounds. Hyperproliferative HaCaT keratinocytes in monolayer cultures were exposed to fumaric acid, dimethylfumarate, zinc monoethylfumarate, calcium monoethylfumarate and magnesium monoethylfumarate at concentrations between 0.4 microM and 960 microM for 48 h. Cell proliferation was studied by [3H]thymidine incorporation. In addition 14C-labelled amino acid uptake and total protein content were measured. Direct cytotoxicity was determined by the release of cytoplasmic lactate dehydrogenase (LDH) into the culture medium. The corresponding 50% inhibition concentrations (IC50) were calculated for DNA/protein synthesis: 2.3/2.5 microM (dimethylfumarate), 133/145 microM (zinc monoethylfumarate), 215/230 microM (calcium monoethylfumarate), 275/270 microM (magnesium monoethylfumarate), > 960/> 960 microM (fumaric acid). The total protein content was less sensitive. Antiproliferative activity was found for dimethylfumarate and to a lesser degree for calcium monoethylfumarate already at the subtoxic concentrations of 1.3 and 4 microM, respectively. In the case of magnesium monoethylfumarate, zinc monoethylfumarate and fumaric acid there was no such dissociation between their cytotoxic and antiproliferative potential. These data indicate that most of the antipsoriatic potential of fumaric therapies is due to the dimethylfumarate compound.
Cell Death↗
IL-1 alpha-induced expression of ICAM-1 on cultured hyperproliferative keratinocytes: suppression by antipsoriatic dimethyl-fumarate.
BACKGROUND: In the psoriatic plaque both IL-1 dysregulation and ICAM-1 expression on keratinocytes have been previously described. Furthermore systemic administration of fumarates has been reported to be effective in psoriasis. We, therefore, studied the effect of dimethyl-fumarate ester (DMF) on the putative IL-1-induced ICAM-1 expression. METHODS: Hyperproliferative human keratinocytes (HaCaT cell line) were incubated in 10 to 100 U/mL IL-1 alpha for 24 h with and without preincubation with 0.4-12.0 microM DMF: Expression of ICAM-1 was measured by a special ELISA-APAAP technique. RESULTS: The exposure to IL-1 led to a significant dose-dependent induction of ICAM-1 expression of from 124 +/- 17 to 194 +/- 22% (control 100 +/- 12%), while proliferation remained unaltered. Pretreatment with > or = 4 microM DMF resulted in a distinct suppression of ICAM-1 expression and a slight decrease in proliferation. CONCLUSIONS: The present results show that ICAM-1 expression on hyperproliferative keratinocytes may be triggered by IL-1 alpha and serve as a molecular target for antipsoriatic DMF.
Cell Adhesion Molecules↗
Anthralin: how does it act and are there more favourable derivatives?
Anthralin is still the most effective and safest therapeutic agent for treatment of psoriasis. Our data may assist toward an understanding of its mode of action and introduce new derivatives, more antiproliferative and less toxic than anthralin in vitro. Anthralin exerts a direct effect on keratinocytes and leukocytes. In time-lapse studies it significantly prolonged the prophase of mitotic keratinocytes in subtoxic doses and suppressed the expression of keratin 6 mRNA in the immediately suprabasal layer of psoriatic epidermis in vivo. Anthralin inhibits the transformation of lymphocytes and the release of reactive oxygen species from activated leukocytes, in vitro. We provide evidence that these effects of anthralin are mediated by protein kinase C. Twelve new hydrophilic derivatives of anthralin, including a 1,8-dimethoxy compound, as well as C-2 and C-10 substituted anthrones were tested on human keratinocytes. The antiproliferative effect of those derivatives bearing lacton rings at a C-10, consisting of 4, 5, or 6 C atoms, exceeded that of anthralin and were equally or less cytotoxic than the parent drug. These compounds had no pro-drug character in vitro, since they did not metabolize via anthralin, as shown by HPLC. These data indicate that there may be anthralin derivatives with more favourable properties for topical therapy than anthralin itself.
Anthralin↗
[An anaphylactic shock reaction caused by latex from medical protective gloves].
In case of IgE-mediated latex allergy, the person wearing medical rubber gloves as well as the patient getting in contact with the gloves' outside may be affected. This is illustrated by the present case reports on two atopic young women. The first patient repeatedly underwent laparotomies and finally, 50 minutes after the onset of another operation, suffered an anaphylactic shock reaction due to transperitoneal latex resorption. The second patient was trained as an assistant in a dentist practice where she suffered more than once from latex contact urticaria as well as collapses. Since these were initiated by globus feeling, an aerogenic route of allergen penetration was assumed. Latex prick testing in this patient resulted in an anaphylactic shock reaction. With regard to these observations the general aspects of intraoperative allergen exposition and safety of latex skin testing are discussed. Recommendations are given for a) the latex test procedure in case of anamnestic suspicion of high-degree sensitisation, b) the issuing of a latex allergy pass, and c) the use of latex-free synthetic gloves.
Adolescent↗
Keratin pattern of acanthosis nigricans in syndromelike association with polythelia, polycystic kidneys, and syndactyly.
BACKGROUND: Acanthosis nigricans (AN) comprises a broad spectrum of etiologic subtypes. The underlying pathomechanisms have not yet been completely clarified. We present a patient affected with a syndromelike AN subtype including disturbed epidermopoiesis as evidenced by immunohistologic findings and in situ hybridization. OBSERVATIONS: A 54-year-old white man contracted AN during childhood. There were connate malformations consisting of webbed toes II/III on the right side and a supernumerary left mammilla. As an adult he developed psoriasis vulgaris, obesity, and latent diabetes mellitus, polycystic kidney and liver disease. With regard to keratin 6 mRNA, and the protein expression of keratin 6/16, KI-67, and proliferating cell nuclear antigen, the AN lesion showed moderate hyperproliferation. A much higher degree of hyperproliferation was evident in psoriatic areas of the patient's skin. In contrast to psoriatic tissue, basal keratinocytes of the AN showed an unusually high expression of keratin 18 and 19 protein. CONCLUSIONS: The observation thus deals with a unique, syndromelike constellation of AN characterized by a particular epidermal pattern of moderate hyperproliferation. A further dysregulation of protein expression in the epidermis is indicated by the demonstration of the rare keratins 18 and 19 in basal keratinocytes of the AN lesion.
Abnormalities, Multiple↗
Safety of latex prick skin testing in allergic patients.
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Adolescent↗
Interferon-gamma regulates expression of a novel keratin class I gene.
Interferon (IFN)-gamma has been implicated in the pathogenesis of several autoimmune disorders and inflammatory skin diseases. To identify novel mediators involved in the IFN-gamma response we have used differential hybridization of a cDNA library prepared from IFN-gamma-treated HeLa cells to isolate a gene that is induced following treatment with IFN-gamma. We report here the molecular cloning and characterization of a cDNA detecting a 1.6-kb mRNA that accumulated in response to IFN-gamma but not in response to IFN-alpha or IFN-beta. The gene is regulated by IFN-gamma in human cell lines of epithelial origin. The mRNA encodes a predicted protein of 432 amino acids and the primary structure of the protein demonstrates that it is a novel member of developmentally regulated keratin class I genes.
Amino Acid Sequence↗
Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.
Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.
Alkaloids↗
Anti-proliferative effects of protein kinase C inhibitors in human keratinocytes.
Various lines of evidence indicate that protein kinase C, a key enzyme in transmembraneous signal transduction, is involved in the regulation of keratinocyte proliferation. In the present study we have investigated the effects of various structurally unrelated protein kinase C inhibitors on the proliferation of HaCa T cells, a non-tumorigenic human keratinocyte cell line. All protein kinase C inhibitors dose-dependently inhibited cell proliferation as assessed by the incorporation of radioactively labelled thymidine and amino acids as well as the increase in total protein content in keratinocytes. The potencies of the drugs to inhibit cell proliferation were strongly correlated to their inhibitory potency on purified protein kinase C, displaying a correlation coefficient of 0.97. Methotrexate, an anti-proliferative drug, was found not to inhibit protein kinase C. Therefore, our data provide evidence that protein kinase C is crucially involved in the regulation of keratinocyte proliferation but is not the only target of anti-proliferative drug action.
Cell Division↗