Efficacy and safety of Id-protein-loaded dendritic cell vaccine in patients with multiple myeloma--phase II study results.

UNLABELLED: In a phase II clinical study, pretreated multiple myeloma patients with relapsing or stable disease received autologous anticancer vaccine containing dendritic cells loaded with Id-protein. Patients received a total of 6 vaccine doses intradermally in monthly intervals. No clinical responses were observed. During the follow-up with a median of 33.1 months (range: 11-43 months), the disease remained stable in 7/11 (64%) of patients. Immune responses measured by ELISpot were noted in 3/11 (27%) and DTH skin test for Id-protein was positive in 8/11 (73%) of patients; out of those, 1/11 (9%) and 5/11 (46%), respectively, had preexisting immune response to Id-protein before the vaccination began. Outcomes were compared to those of a control group of 13 patients. A trend to lower cumulative incidence of progression in the vaccinated group was observed at 12 months from the first vaccination (p= 0.099). More patients from the control group compared to vaccinated patients required active anticancer therapy [4/11 (36%) vs. 8/13 (62%)]. Vaccines based on dendritic cells loaded with Id-protein are safe and induce specific immune response in multiple myeloma patients. Our results suggest that the vaccination could stabilize the disease in approximately two-thirds of patients. KEYWORDS: dendritic cells, immunotherapy, anticancer vaccines, Id-protein, multiple myeloma.

Generation of myeloma-specific T cells using dendritic cells loaded with MUC1- and hTERT- drived nonapeptides or myeloma cell apoptotic bodies.

Dendritic cells are able to induce anti-tumor immune responses by presenting tumor-specific antigens to T-lymphocytes. Various tumor-associated antigens have been studied in multiple myeloma in an effort to find a strong antigen capable of generating clinically meaningful responses in vaccinated patients. The aim of our study was to generate myeloma-specific cytotoxic T lymphocytes in vitro using dendritic cells loaded with peptide antigens or apoptotic bodies. Peripheral blood mononuclear cells from HLA-A2+ healthy donors were used for isolation and culture of dendritic cells (DCs) and T lymphocytes. DCs were loaded with hTERT- and MUC1-derived nonapeptides or apoptotic bodies from myeloma cells. Repeated stimulation of T lymphocytes led to their activation characterized by interferon-gamma production. Activated T lymphocytes were separated immunomagnetically and expanded in vitro. Specific cytotoxicity of the expanded T lymphocytes was tested against a myeloma cell line. There was evidence of cytotoxicity for all three types of antigens used for T lymphocyte priming and expansion. No statistically significant differences were observed in T lymphocyte cytotoxicity for any of the antigens. We present a method for the priming and expansion of myeloma-specific T lymphocytes using dendritic cells loaded with different types of tumor antigens. Cytotoxic T lymphocytes and/or activated dendritic cells generated by the described methods can be applied for cellular immunotherapy against multiple myeloma and other malignancies.

Comparison of methods used for evaluation of mutagenicity/genotoxicity of model chemicals - parabens.

Growing worldwide efforts to replace (reduce) animal testing and to improve alternative in vitro tests which may be more efficient in terms of both time, cost and scientific validity include also genotoxicity/mutagenicity endpoints. The aim of the review article was to summarize currently available in vitro testing approaches in this field, their regulatory acceptance and recommended combinations for classification of chemicals. A study using the combination of Comet Assay performed on two cell lines and the Chromosomal Aberration test on human peripheral lymphocytes was performed with the aim to predict the genotoxic potential of selected paraben esters, serving as a model chemical group. Parabens are widely used in consumer products as preservatives and have been reported to exhibit inconclusive results in numerous genotoxicity studies. The Comet Assay identified Ethylparaben and Benzylparaben as potentially genotoxic. The Chromosomal Aberration test revealed weak genotoxic potential in case of Ethylparaben and positive genotoxicity in case of Butylparaben, Propylparaben and Isopropylparaben. The main reasons for variability seem to be limited water solubility of parabens, determining their bioavailability at the cellular level, and absence of metabolic activation in the Comet Assay. The results confirmed that the Comet Assay should serve as a screening test and should not be used as a stand-alone method for classification of genotoxicity. The weight of evidence approach in risk assessment should be supported with data generated with the use of human relevant in vitro methods based on cells / tissues of human origin.

[The preparation of anticancer vaccine for patients with multiple myeloma on the base of monoclonal immunoglobulin loaded dendritic cells]. / Príprava protinádorové vakcíny pro pacienty s mnohocetným myelomem na bázi dendritických bunek nalozených monoklonálním imunoglobulinem.

BACKGROUND: On June 2006, phase II clinical trial focused on anticancer vaccination of multiple myeloma patients, was started. On September 2007, the immune and clinical response evaluation of first four patients was finished.The anticancer vaccine contained dendritic cells loaded with monoclonal immunoglobulin produced by myeloma cells. METHODS AND PATIENTS: Within the frame of phase II clinical trial were vaccinated four myeloma patients with stable disease. It was administered six vaccines for each patient, monthly. The dendritic cells were cultured from the patient's peripheral blood mononuclear cells and loaded with autologous monoclonal immunoglobulin under the good manufacturing practice conditions. After the safety and quality control, the satisfactory vaccine was administered to the patient. The functional characteristic of dendritic cells was evaluated using flow cytometry, the immune response was evaluated using ELISpot. The clinical response was monitored using monoclonal immunoglobulin concentration in patient's sera. RESULTS AND CONCLUSION: The immune response detected using ELISpot was observed in 3/4 patients. The monoclonal immunoglobulin concentration was changeable for all twelve months, but never exceeded the range of 25% for minimal clinical response achievement. During the vaccination, no significant toxicities or negative side-effects were observed. The clinical trial is going on with vaccination other patients with multiple myeloma.

[The preparation of myeloma-specific T cells activated with dendritic cells loaded with nonapeptides derived from mucin protein MUC1 and catalytic subunit of telomerase hTERT]. / Príprava myelom-specifických t lymfocytu aktivovaných dendritickými bunkami nalozenými nonapeptidy odvozenými od mucinového proteinu MUC1 a katalytické podjednotky telomerázy hTERT.

BACKGROUND: Multiple myeloma is an incurable hematological disease. High-dose chemotherapy including autologous stem cell transplantation is recently considered a standard therapy for myeloma. Unfortunately, a relapse of the disease is inevitable. Therefore, new approaches such as immunotherapy have been considered recently. A specific activation of cytotoxic T cells can be reached using dendritic cells loaded with tumor-specific antigens. The HLA-A2-specific nonapeptides as hTERT derived from catalytic subunit of telomerase and MUC1 derived from mucin protein can be used. DESIGN AND SUBJECTS: Activation, identification, separation and expansion of myeloma-specific T cells from healthy HLA-A2 blood donors were tested in an in vitro study using hTERT and MUC1 nonapeptides as tumor-specific antigens. METHODS AND RESULTS: T cells and dendritic cells were obtained from peripheral blood. T cells were repeatedly stimulated with hTERT and MUC1 nonapeptide-loaded dendritic cells. Activated myeloma-specific T cells produced interferon gamma and were evaluated by flow cytometry. The activated T cells were immunomagnetically separated and in vitro expanded to the number usable in clinical trials. CONCLUSIONS: This study demonstrates feasibility of a specific activation, identification, separation and expansion of tumor-specific T cells that can be used in myeloma therapy.

[Selective depletion of alloreactive T cells and study of anti-tumor activity of specific T cell clones in patients with leukemia]. / Selektivní deplece aloreaktivních T lymfocytu a studium protinádorové aktivity specifických klonu T lymfocytu u pacientu s leuikémií.

BACKGROUND: Graft-versus-host disease (GVHD) is a severe complication of allogeneic transplantation of hematopoietic stem cells. Donor T cells play a major role in GVHD leading to the host tissue damage, mainly the skin, liver, and gastrointestinal tract. A selective depletion using an anti-CD25 immunotoxin can eliminate harmful alloreactive T cells while preserving other donor T cells with antileukemic and antiinfectious reactivity. PATIENTS AND METHODS: We performed 15 mixed lymphocyte reactions with clinical specimens from 12 patients with various types of leukemia (7x AML, 3x ALL, 1x CML, 1x CLL) and PBMC from 15 healthy volunteers from Transfusive station FN Brno Bohunice. RESULTS: In our experiments we have demonstrated, that antileukemic (GVL) effect of donor, especially CD4+ T cells was well preserved (7.46%), while unfavourable alloreactive (GVH) reaction of donor T cells was completely removed. The graft-versus-host (GVH) reactivation of donor cells was negligible ever after repeated stimulation with irradiated patient's PBMC. CONCLUSION: We have shown that anti-CD25 immunotoxin (IT), RFT5-SMPT-dgA, launched against alpha chain for human interleukin 2 (IL-2), led to long-term selective depletion of alloreactive donor T cell clones while their antileukemic activity was well preserved. Base on our results the clinical phase I/II study was designed. This study was initiated in year 2007 in three clinical centers in Czech Republic.

Dendritic cell counts and their subsets during treatment of multiple myeloma.

Human dendritic cells have distinct roles in the regulation of immunity. In this study we analysed the kinetics and the proportion of myeloid and plasmacytoid subsets of dendritic cells (DC) in peripheral blood of 15 patients with multiple myeloma (MM) before and during treatment that included autologous transplantation. Control group of 15 healthy volunteers was evaluated by using the same approaches. Flowcytometric determination of relative and absolute cell counts in unmanipulated peripheral blood was based on the expression of surface antigens CD83 and HLA-DR. Depending on the expression of CD11c or CD123, we divided these cells into CD11c+ dendritic cells type 1 (DC1) and CD123+ DC type 2 (DC2). Significant differences were found in initial relative counts of CD83+ cells and of the DC2 subtype between the group of controls and the group of patients before treatment. In absolute counts, there was a difference only in the DC2 subtype. After induction treatment (vincristine, doxorubicin, and dexamethasone), the mean percentage of CD83+ DC and the DC1 percentage were significantly higher than initially, but there was no significant difference in absolute counts. Administration of G-CSF again increased the total DC numbers. Intermediate DC counts were found in the apheresis products. After engraftment, we found the highest relative DC numbers, but absolute counts were not very high because of leukopenia. Within six months after transplantation, normal relative and absolute DC counts were found in patients. Untreated patients with MM have significantly lower relative numbers of peripheral blood DC in comparison with healthy volunteers. The highest number of total DC was found after engraftment. The DC1/DC2 ratio showed relative predominance of DC1 subtype and the lowest DC1/DC2 ratio was found in the apheresis products. DC counts comparable with those of healthy volunteers were found in patients six months after transplantation.

Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style.

The original purpose of our study was to determine if the detection of chromosomal aberrations in peripheral lymphocytes of children might be used as a biomarker of environmental pollution and life style. We compared the results of cytogenetic analyses performed in children and adolescents in the periods 1984-1993 and 1994-1999, in a total of 3402 subjects. The frequency of aberrant cells (AB.C.) markedly decreased in the period 1994-1999 compared with the period 1984-1993. The decreases in AB.C. were significant in the age groups 7-15 and 16-19 years: 1.63% AB.C. versus 1.14% AB.C. and 2.02% AB.C. versus 1.08% AB.C., respectively (P<0.01). No difference in the frequency of AB.C. was observed in newborns. Based on our experience, we believe that monitoring the spontaneous level of chromosomal aberrations in children over 5 year periods may be used to examine the general changes in environmental pollution in larger geographic areas.

Genotoxicity of industrial effluents, river waters, and their fractions using the Ames test and in vitro cytogenetic assay.

Total and fractionated organic extracts from industrial effluents and Labe river water were tested for mutagenicity using the Ames test with TA98 strain and its YG derivatives (YG1021, YG1024 and YG1041) and cytogenetic analysis with human peripheral lymphocytes in vitro. The bacterial mutagenicity results showed a dose-dependent increase in numbers of TA98 revertants (10(5)) with effluent extracts and lower, but still significant, increase (10(3)) with river water extracts 6 km downstream. The further increase of YG revertants indicates the possible presence of nitroarenes and aromatic amines in the tested samples. In fractionated samples the significant mutagenicity was detected in two-polar acidic and polar basic-fractions, but the numbers of revertants in all effluent fractions were about one order of magnitude lower compared with total extracts results. The cytogenetic effect in human peripheral lymphocytes in vitro was not significant. The increase in chromosomal aberrations was not clearly dose-dependent and may reflect more the combination of toxic and clastogenic effects.

Spontaneous level of chromosomal aberrations in peripheral blood lymphocytes of control individuals of the Czech Republic population.

In order to assess the potential of cytogenetic determinations on peripheral blood lymphocytes as a mean of monitoring human population subjects to occupational and environmental exposures to genotoxins, accurate baseline data are required. During the past 20 years many results of the cytogenetic studies on peripheral blood lymphocytes from monitored occupationally exposed and non-exposed groups were obtained. At the time of blood drawing a questionnaire was administered. The questions covered a brief medical and family history including age, sex, medication, infectious diseases, smoking habits, X-ray examinations, alcohol consumption etc. Cytogenetic analysis from whole blood was carried out in short-term cultures. The cultivation time was 52 hours with all cells being in the first mitosis. A total of 100 well-spread metaphases containing 46 +/- 1 centromere were examined per donor on coded slides. Four categories of chromosome aberrations were evaluated: Chromatid and chromosome breaks, chromatid and chromosome exchanges. Cells bearing breaks or exchanges were classified as aberrant cells. Gaps were recorded but not scored as aberrations. Results of the cytogenetic analysis from control individuals (N = 5,430) indicated elevation of spontaneous frequency of aberrant cells (AB.C.) with age. We found 1.10% AB.C. (N = 551) in newborns; 0.71% AB.C. (N = 105) in the group 5-6 yr; 1.20% (N = 1,734) in the group 7-15 yr; 1.25% AB.C. (N = 239) in the group 16-19 yr and 1.59% (N = 2,801) in the group 20-63 yr.

Population-based biomonitoring in the Czech Republic--the system and selected results.

In the framework of the system of monitoring the environmental impact on population health, the concentration of lead, cadmium and selenium in blood and cadmium in urine was measured in adults (n = 670), children (n = 599) and umbilical blood (n = 549) using atomic absorption spectrophotometry. Furthermore, cytogenetic analysis of peripheral lymphocytes in all population groups under study was investigated. The median blood Pb level for the overall group of adults (47.8 micrograms/l, i.e. 0.23 mumol/1) was significantly higher in men (51.5 micrograms/l, i.e. 0.25 mumol/l). Smoking significantly influenced the blood Pb level in women. The 90th percentile in no group exceeded the value of 100 micrograms/l (0.48 mumol/l). The median blood Cd level in adults (0.9 microgram/l, i.e. 0.008 mumol/l) depends on smoking habit (1.25 micrograms/l, i.e. 0.01 mumol/l). The median urine Cd level was 0.585 microgram/g creatinine (0.59 mumol/mole creatinine) in adults and 0.37 microgram/g creatinine (0.37 mumol/mole creatinine) in children. The median blood Se level (53.5 micrograms/l, i.e. 0.68 mumol/l) was found to be higher in the group of non-smokers (57.5 micrograms/l, i.e 0.73 mumol/l). Lead and selenium level were significantly lower in the umbilical blood. Cytogenetic analysis results showed age-dependent average percentages of aberrant cells: 1.1% in umbilical blood, 1.27% in children and 1.71 in adults in line with the reference values for the Czech population.

Monitoring of human exposure to occupational genotoxicants.

The human exposure to genotoxic agents can be detected by using genetic monitoring procedures which is mainly concerned with markers of exposure and effect. Cytogenetic analysis of human peripheral lymphocytes and urine mutagenicity are routinely used in Hygiene Service of the Czech Republic. The review demonstrated the activity of National Reference laboratory and other laboratories in Hygiene Service of the Czech Republic in the problem dealing with monitoring of population exposure to genotoxic substances. Altogether, 7 regional and 15 district laboratories have been in function. Several thousands of occupationally exposed and control persons have been examined by now. The most followed population at risk were those exposed to cytostatics, polycyclic aromatic hydrocarbons, complex mixtures of chemicals, metals and others. The system of genetic monitoring helped to detect the exposure of population at risk to genotoxic contaminants, to use the obtained data for quantification of exposure and for preventive measures application and to control the efficacy of applied regulatory action.

Induction of regulatory T Cells by dendritic cells through indoleamine 2,3-dioxygenase: a potent mechanism of acquired peripheral tolerance.

Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway. Recent works have demonstrated a crucial role for IDO in the induction of immune tolerance during infections, pregnancy, transplantation, autoimmunity, and neoplasias. IDO is widely expressed in human tissues and cell subsets, including dendritic cells, where it modulates their function by increasing tolerogenic capacities. The aim of the present paper is to highlight the most recent data about IDO expression in dendritic cells and its role as a potent inducer of T regulatory cells.

Generation of dendritic cells using cell culture bags--description of a method and review of literature.

Anticancer immunotherapy using dendritic cell-based vaccines is a strategy aimed at the induction and maintenance of immune responses against cancer cells. Clinical applications of dendritic cells (DCs) require stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization of DC-based vaccine preparation. Recently, closed systems for DC culture have been developed with a goal to minimize the risk of contamination. Here, we compare the yield, immunophenotype, and functional properties of DCs generated in Lifecell X-Fold culture bags and in plastic wells, both from adherence-selected monocytes, and review the current literature on closed systems for DC generation. We found that both the overall yield and the yield of CD83+ cells in cell culture bags was lower than in the standard culture method. No statistically significant differences were observed in the expression of DC immunophenotypic markers. The capability of DCs cultured in bags and in wells to induce the proliferation of allogeneic mononuclear cells were equivalent. The performance of DCs in mixed lymphocyte reaction correlated significantly (p = 0.005) with the CD83 expression but not with the CD80, CD86, HLA-DR, CD1a, and CD1c expression. We conclude that the immunophenotype and stimulatory properties of DCs cultured in closed cell culture bags are similar to those generated by conventional method using cell culture wells.