Skin exposure to deodorants/antiperspirants in aerosol form.

Many cosmetic products are available in spray form. Even though the principal targets of these products are the skin and hair, spraying leads to the partitioning of the product between the target and the surrounding air. In the previous COLIPA study (Hall et al., 2007) the daily use of deodorant/antiperspirant (Deo/AP) in spray form was quantified in terms of the amount of product dispensed from the spray can, without specifically quantifying the product fraction reaching the skin during use. Results of the present study provide this additional information, necessary for a reliable safety assessment of sprayed Deo/AP products. In a novel experimental approach the information obtained from real-life movement analysis (automated motion imaging) of volunteers using their own products was integrated with the aerosol cloud sampling data obtained from the same products, leading to the computation of the product deposited on the skin. The 90th percentile values, expressed as percent deposition relative to the can weight loss after spraying, are 23.5% and 11.4% for ethanol-based and non-ethanol-based products, respectively. Additionally, the study has generated data on the skin area covered by the products, spray duration time, spray angle and spray distance from the skin.

Enhanced lytic activity of cytokine-induced killer cells against multiple myeloma cells after co-culture with idiotype-pulsed dendritic cells.

BACKGROUND AND OBJECTIVES: There are numerous reports of in vitro and in vivo usage of dendritic cells (DC) pulsed with idiotype, the tumor-specific antigen of multiple myeloma (MM), for immunotherapy of MM. Data suggest that not only T-cells, but also the innate immune system reacts against MM. Here, we examined the cytotoxic activity of cytokine-induced killer (CIK) cells against myeloma cells. This heterogeneous effector population consists of T-, NK- and NKT-cells. DESIGN AND METHODS: CIK cells generated from buffy coats or blood from patients with MM were co-cultured with autologous idiotype-pulsed DC. The cytotoxic activity was investigated in lactate dehydrogenase release assays against cell lines or autologous CD138 positive cells from bone marrow. RESULTS: CIK cells were able to lyse MM cells at low effector to target ratios. This effect was significantly enhanced by co-culturing with specifically pulsed DC (83.8% lysis at an effector to target ratio of 16:1). Using an interferon-g secreting MACS separation assay, the cytotoxic activity of CIK cells was enhanced to maximal lysis at the lower effector to target ratio of 5:1. High cytotoxic activity was also shown in a completely autologous setting against enriched CD138+ cells from a patient with MM (54.4% lysis at an effector to target ratio of 6:1). Interestingly, there was no cytotoxic activity against the CD138- fraction of the bone-marrow. INTERPRETATION AND CONCLUSIONS: Using a heterogeneous population of effector cells, we were able to activate the innate and the adoptive immune-system against myeloma cells. CIK cells showed high lytic activity against MM cells, which could be enhanced by co-culturing with antigen-specific pulsed DC.

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells.

The present study assessed the role of adenoviral vector-mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines (n=5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53-transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-beta-Gal-transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols.

A novel minimal-size vector (MIDGE) improves transgene expression in colon carcinoma cells and avoids transfection of undesired DNA.

Viral and plasmid vectors may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes contained in their sequence. Furthermore, replication-defective viral vectors carry the potential risk of recombination with wild-type viruses or activation of oncogenes. A new vector type for minimalistic, immunologically defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal-size gene transfer unit containing the expression cassette, including promoter, gene, and RNA-stabilizing sequence, flanked by two short hairpin oligonucleotide sequences. The resulting vector is a small, linear, covalently closed, dumbbell-shaped molecule. DNA not encoding the desired gene is reduced to a minimum. Here, we transfected colon carcinoma cell lines using cationic lipid, cationic polymer, and electroporation with several MIDGE vectors and corresponding plasmids containing transgenes encoding enhanced green fluorescent protein (eGFP) and human interleukin-2 (hIL-2). Transfection efficiency as measured qualitatively and quantitatively with eGFP was found to be comparable for both vector types. However, hIL-2 secretion and eGFP expression were approximately two- to fourfold higher in most cells transfected with these transgenes using MIDGE vectors compared to the plasmid control. This report demonstrates the advantages of this new vector type and its prospects for ex vivo gene therapy studies.

Transfection of dendritic cells (DCs) with the CIITA gene: increase in immunostimulatory activity of DCs.

Dendritic cells (DCs) are the major antigen-presenting cells. They are able to present tumor antigens to immunologic effector cells. MHC class II molecules on DC surfaces play an important role in priming effector cells against tumor cells and their antigens. The transactivator CIITA (MHC class II transactivator) is a non-DNA-binding transactivator, which regulates the expression of MHC class II, HLA-DM, and invariant chain and behaves as a master controller of constitutive and inducible MHC class II gene activation. Here, we transfected DCs with the CIITA gene using a novel transfection technique. The vector system consisted of a plasmid bound to an adenovirus via poly-L-lysine, which is covalently bound to a UV-irradiated adenovirus. After transfection, expression of MHC class II on DCs increased from 27% to 75% on day 2 after transfection. Transfected DCs were co-cultured with immunologic effector cells. Cytotoxicity of effector cells against tumor cells increased after co-culture with transfected DCs to 63% compared to 15% with effector cells co-cultured with irrelevantly transfected DCs (P=.037). This effect was dependent on the timing and period of co-culture. In conclusion, transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs. We can further conclude that DCs could be efficiently transfected with the CIITA gene. Transfection of DCs led to an increase in antitumoral immunostimulatory capacity of DCs and may have a major impact on immunotherapeutic protocols for patients with cancer.

Resistance of pancreatic carcinoma cells is reversed by coculturing NK-like T cells with dendritic cells pulsed with tumor-derived RNA and CA 19-9.

Immunization with defined tumor antigens is limited to the small number of cancers in which specific tumor antigens have been defined but insufficient tumor material is available to produce an antitumor vaccine. In this study, we investigated whether pulsing dendritic cells (DC) using a liposomal transfer technique with a pancreatic tumor cell line-derived RNA can effectively activate NK-like T cells and tumor immunity. Pulsed DC were cocultured with NK-like T cells, i.e., CD3+CD56+ cells, as immunologic effector cells. Target cells resistant to NK-like T-cell-mediated lysis were used. Total tumor-derived RNA transfected into DC was found to completely reverse tumor cell resistance. Total tumor RNA transfection (30 microg) was found to be superior to poly(A)(+) RNA transfection (5 microg) in inducing NK-like T lymphocytes. Interestingly, additional pulsing of DC with the CA 19-9 peptide in a CA 19-9-positive cell line further increased the sensitivity of pancreas carcinoma cells to NK-like T cells. Treatment of tumor RNA with RNase completely blocked the effect of RNA-transfected DC on NK-like T cells, suggesting that intact tumor-derived RNA is needed for reversal of tumor cell resistance. In conclusion, coculture of NK-like T cells with DC transfected with pancreatic tumor cell line-derived RNA reverses pancreatic tumor cell resistance by directly triggering NK-like T lymphocytes.

The Epstein-Barr virus latent membrane protein 1 induces interleukin-10 in Burkitt's lymphoma cells but not in Hodgkin's cells involving the p38/SAPK2 pathway.

Infection of B cells with Epstein-Barr Virus (EBV) induces interleukin-10 (IL-10) production, which may contribute to transformation. IL-10 can modulate the immune response at certain levels, playing a crucial role in balancing humoral and cellular responses. Moreover, it can function as a growth and differentiation factor for B cells. However, the mechanism of IL-10 induction is still unclear. Here we demonstrate that IL-10 was specifically induced by the EBV-latent membrane protein 1 (LMP1) in Burkitt's lymphoma (BL) cell lines BL2 and BL41. In two T cell lines (Jurkat, MOLT3), two NHL cell lines (U266, MHH-PREB1), or three Hodgkin's disease (HD) cell lines (L428, L540, and KMH2), LMP1 did not induce IL-10 expression. In contrast, LMP1 activated CD40 or CD54 (ICAM1) expression in the analyzed cell lines. LMP1 derivatives lacking the C-terminal activation regions (CTAR), by deletion of the amino acids between 187 and 351 (Delta CTAR1) or 232 and 386 (Delta CTAR2), alone, or together induced IL-10 at very low amounts compared to wild-type LMP1. Inhibition of LMP1-mediated NF kappa B activation by constitutive repressive I kappa B-alpha only marginally impaired IL-10 expression in BL2 cells, while SB2035080 at 5 microM (a specific p38/SAPK2 inhibitor) led to reduced IL-10 expression. Our findings confirm the role of LMP1 in transactivation of cellular genes possibly important for tumor immunoescape but show that more than one signaling pathway is involved in this activation and suggests the necessity of a defined conformation of CTARs to activate IL-10 involving p38/SAPK2.

Interactions between dendritic cells and cytokine-induced killer cells lead to an activation of both populations.

Dendritic cells (DCs) are major antigen-presenting cells. They are capable of capturing and processing tumor antigens, expressing lymphocyte costimulatory molecules, and secreting cytokines to initiate immune responses. Here, the authors tested the effect of cytokine-induced killer (CIK) cells, a population that includes CD3+CD56+ cells (natural killer T cells), with regard to their capacity to immunomodulate DCs. Cytokine-induced killer cells were cocultured with autologous DCs generated from peripheral blood mononuclear cells. Expression of markers typical for both populations was measured using flow cytometry, and secretion of interleukin (IL)-12 was determined using enzyme-linked immunosorbent assays. Cytotoxicity assays were performed to investigate the role of IL-12 and the importance of cell-cell interactions. Considering this, receptors for IL-12 and CD40 were blocked and cocultures were performed with cell culture inserts. Coculture of CIK cells led to a significant increase of DC-specific, costimulatory, and antigen-presenting molecules in DC cultures. In addition, coculture resulted in a dramatically increase of IL-12 secretion by DCs and to a significant increase in cytotoxic activity of CIK cells toward carcinoma cells. Blockage of IL-12 uptake decreased the cytolytic activity of CIK cells. Cytokine secretion was shown to be important for activation of CIK cells, and also cellular interactions between DCs and effector cells caused a higher cytolytic capacity. Interactions between DCs and CIK cells caused changes in the surface molecule expression of both populations, led to an increase of IL-12 secretion, and rendered an improved cytotoxic activity. The natural killer T cell subpopulation seems to be responsible for this effect. Therefore, coculture of DCs with CIK cells may have a major impact on immunotherapeutic protocols for patients with cancer.

Increase in proliferation rate and normalization of TNF-alpha secretion by blockage of gene transfer-induced apoptosis in lymphocytes using low-dose cyclosporine A.

Efficient gene transfer of lymphocytes is extremely difficult. We have shown previously that induction of apoptosis may play a role in the gene transfer resistance of lymphocytes. Anti-CD3 antibody can be used as a surrogate for receptor-mediated gene transfer in T lymphocytes. However, anti-CD3 antibody has been shown to be the causative agent of apoptosis in receptor-mediated gene transfer. In this study, we show that blockage of apoptosis by addition of low-dose cyclosporine A can lead to normalization of elevated TNF-alpha secretion and to a significant increase in the proliferation rate of transfected lymphocytes. In contrast, this had no negative effect on cytotoxic activity of immunologic effector cells called cytokine-induced killer cells. Therefore, blockage of apoptosis should have an impact on the use of lymphocytes transfected with cytokine genes as immunologic effector cells in cancer gene therapy protocols.

Increase in the immunostimulatory effect of dendritic cells by pulsing with serum derived from pancreatic and colorectal cancer patients.

Both we and others have observed a relative resistance of solid tumor cells to immunological effector cells in vitro, which may be one reason for the clinical phenomenon of resistance of patients with pancreatic carcinoma or other solid tumors to immunological therapeutic approaches. Dendritic cells (DC) are professional antigen-presenting cells which can process and present tumor-associated antigens such as CA 19-9. Here we tested DC pulsed with serum containing CA 19-9 for their capacity to stimulate immunological effector cells against pancreatic carcinoma cells. Coculture of immunological effector cells with DC led to a significant increase in cytotoxic activity as measured by a lactic dehydrogenase release assay. Most interestingly, cytotoxic activity against tumor cells was further increased using DC pulsed with patient-derived CA 19-9 containing serum. Similar results have been obtained using either autologous or allogeneic serum from patients with pancreas carcinoma. The effect of serum on the cytotoxicity of effector cells increased in a dose-dependent manner. Interestingly, heat inactivation led to a significant loss of immunostimulatory capacity of the serum. Cytotoxicity was partially inhibited by using an antibody directed against CA 19-9 on the surface of the target cells. Best results were obtained when adding CA 19-9 protein to CA 19-9 containing serum for pulsing of DC. In conclusion, DC pulsed with CA 19-9 containing serum increased the cytotoxic activity of immunological effector cells against pancreatic cancer cells. DC pulsed with CA 19-9 containing serum with or without additional exogenous CA 19-9 protein may have an impact on immunotherapeutic protocols for patients with CA 19-9 secreting tumors.

Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and primary lymphoma cells can be efficiently transfected using an adenoviral vector system. By adding cationic liposomes, the efficiency of adenoviral gene transfer to primary tumor cells could be further improved. This protocol may have an impact on the use of lymphoma cells in cancer gene therapy.

Increase of the immunostimulatory effect of dendritic cells by pulsing with CA 19-9 protein.

Previously, a relative resistance of solid tumor cells to immunologic effector cells was shown in vitro. This resistance could be one reason for the clinical phenomenon of resistance of patients with colon carcinoma or other solid tumors to immunologic therapeutic approaches. In this study, dendritic cells (DCs) pulsed with CA 19-9 protein were tested for their immunostimulatory capacity of immunologic effector cells against cells derived from colon and pancreatic carcinoma. Dendritic cell cultures coexpressed CMRF-44 and CD1a, markers typical of DCs, in 31.5% +/- 5.3% after 13 days of culture. Coculture of NK-like T lymphocytes with DCs led to a significant increase in cytotoxic activity, as measured using a lactate dehydrogenase release assay. Cytotoxic activity could be further increased using DCs pulsed with CA 19-9 protein. The effect of CA 19-9 on increasing the cytotoxic effect of NK-like T lymphocytes was dose dependent. Similarly, cocultivation of DCs with NK-like T cells derived from patients with metastatic pancreatic cancer and elevated CA 19-9 serum levels led to a significant increase in cytotoxic activity. In conclusion, DCs pulsed with CA 19-9 protein can increase the cytotoxic activity of immunologic effector cells against colon carcinoma and pancreatic cancer cells. Dendritic cells pulsed with CA 19-9 protein may have an important effect on immunotherapeutic protocols for patients with cancer.

Targeting of natural killer-like T immunologic effector cells against leukemia and lymphoma cells by reverse antibody-dependent cellular cytotoxicity.

Recently, highly efficient natural killer-like T immunologic effector cells called cytokine-induced killer (CIK) cells have been described. Most interestingly, CIK cells have been shown to eradicate established human lymphoma cells in a severe combined immunodeficient (SCID) mouse xenograft model in vivo. The current study was aimed at increasing the sensitivity of leukemia and lymphoma cells to CIK cells. In particular, the authors wanted to target CIK cells to leukemia and lymphoma cells via reverse antibody-dependent cellular cytotoxicity. Binding of an anti-CD3 monoclonal antibody to CIK cell cultures derived from patients with lymphoma was shown using flow cytometric analysis. For the target side, several B-cell lines were found to express CD19 on the cell surface. There was an impressive increase in sensitivity to CIK-mediated lysis of various lymphoma and leukemia cell lines by preincubation of the targets with a monoclonal antibody against CD3. This increase could be partially blocked by preincubation with anti-CD16 (Fc receptor III) and anti-CD32 (Fc receptor II) antibodies. These data suggest that the increase in cytotoxic activity is caused by Fc receptor-mediated antibody binding. Cytotoxic activity could be further increased by adding an anti-CD28 antibody in addition to anti-CD3. Finally, there was a further increase in sensitivity to CIK-mediated lysis of CD19+ malignant cells using the bispecific OKT3xHD37 antibody with specificity against CD3 and CD19. Interestingly, preincubation of malignant cells with an anti-CD3 monoclonal antibody followed by addition of the bispecific OKT3xHD37 antibody led to a further increase of cytotoxic sensitivity compared with the addition of the bispecific antibody alone. In conclusion, these data suggest that cytotoxic activity of immunologic effector cells can be increased not only by using the bispecific antibody OKT3xHD37 in vitro but also by preincubation of CD19+ leukemia and lymphoma cells with a monoclonal antibody against CD3. In addition, the immunostimulatory effect of the bispecific antibody OKT3xHD37 can be further increased by adding a monoclonal antibody against CD3.

TNF-alpha secretion and apoptosis of lymphocytes mediated by gene transfer.

Efficient gene transfer of lymphocytes is extremely difficult. Apoptosis may play a role in this gene transfer resistance of lymphocytes. Here we show that transfection of lymphocytes via non-viral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via tumor necrosis factor d (TNF-alpha) and the TNF-alpha receptor pathway, we studied the amount of TNF-alpha secreted by lymphocytes transfected without gene insert. TNF-alpha secretion was dependent on the gene transfer method used. High amounts were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF-alpha were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF-alpha secretion was due to the use of anti-CD3 antibody. Transfection of lymphocytes led to selective decrease in CD120b/TNF-alpha receptor II (TNFR-2)-positive cells. Induction of apoptosis and necrosis mediated by TNF-alpha via TNFR-2 (p80) was partially blocked using a neutralizing anti-TNF-alpha antibody. Blockage of apoptosis and necrosis could be further increased by adding anti-Fas-ligand (FasL) antibody, suggesting that induction of apoptosis via FasL and Fas receptor (Apo-1/CD95) may also play a role. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with cytokine genes. In conclusion, various gene transfer techniques led to TNF-alpha secretion, apoptosis and necrosis of lymphocytes. Apoptosis and necrosis could be partially blocked using a neutralizing anti-TNF-alpha antibody.