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.

• MeSH
• alternativy testů na zvířatech * MeSH
• buněčné linie keratinocytů HaCaT MeSH
• chromozomální aberace chemicky indukované   MeSH
• hodnocení rizik MeSH
• kometový test MeSH
• lidé MeSH
• lymfocyty účinky léků   patologie   MeSH
• mikrojaderné testy MeSH
• mikrojádra chromozomálně defektní chemicky indukované   MeSH
• mutageneze účinky léků   MeSH
• parabeny toxicita   MeSH
• poškození DNA * MeSH
• testy genotoxicity * MeSH
• viabilita buněk účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• srovnávací studie MeSH
• Názvy látek
• parabeny MeSH

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.

• MeSH
• adjuvancia imunologická MeSH
• dendritické buňky imunologie   transplantace   MeSH
• ELISA MeSH
• hemokyanin imunologie   MeSH
• imunoterapie * MeSH
• inhibitory diferenciace imunologie   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• míra přežití MeSH
• mnohočetný myelom imunologie   terapie   MeSH
• pozdní přecitlivělost MeSH
• prognóza MeSH
• protinádorové vakcíny terapeutické užití   MeSH
• senioři MeSH
• staging nádorů MeSH
• studie případů a kontrol MeSH
• vakcinace MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze II MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• adjuvancia imunologická MeSH
• hemokyanin MeSH
• inhibitory diferenciace MeSH
• protinádorové vakcíny MeSH

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.

• MeSH
• aktivace lymfocytů MeSH
• apoptóza * MeSH
• cytotoxické T-lymfocyty imunologie   MeSH
• dendritické buňky imunologie   MeSH
• imunoterapie adoptivní MeSH
• interferon gama biosyntéza   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mnohočetný myelom imunologie   patologie   terapie   MeSH
• mucin 1 imunologie   MeSH
• peptidové fragmenty imunologie   MeSH
• telomerasa imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• interferon gama MeSH
• MUC1 protein, human MeSH Prohlížeč
• mucin 1 MeSH
• peptidové fragmenty MeSH
• telomerasa MeSH
• TERT protein, human MeSH Prohlížeč

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.

• MeSH
• dendritické buňky imunologie   MeSH
• idiotypy imunologie   MeSH
• lidé MeSH
• mnohočetný myelom imunologie   terapie   MeSH
• monoklonální protilátky imunologie   MeSH
• protinádorové vakcíny terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• klinické zkoušky, fáze II MeSH
• Názvy látek
• idiotypy MeSH
• monoklonální protilátky MeSH
• protinádorové vakcíny MeSH

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.

• MeSH
• buněčné klony MeSH
• dítě MeSH
• dospělí MeSH
• imunokonjugáty MeSH
• leukemie imunologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfocytární deplece * MeSH
• mladiství MeSH
• mladý dospělý MeSH
• monoklonální protilátky MeSH
• nemoc štěpu proti hostiteli imunologie   prevence a kontrola   MeSH
• receptor interleukinu-2 - alfa-podjednotka imunologie   MeSH
• ricin MeSH
• T-lymfocyty imunologie   MeSH
• test smíšené lymfocytární kultury MeSH
• transfuze lymfocytů MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• imunokonjugáty MeSH
• monoklonální protilátky MeSH
• receptor interleukinu-2 - alfa-podjednotka MeSH
• RFT5-SMPT-dgA immunotoxin MeSH Prohlížeč
• ricin MeSH

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.

• MeSH
• aktivace lymfocytů MeSH
• antigeny nádorové imunologie   MeSH
• dendritické buňky imunologie   MeSH
• epitopy MeSH
• imunoterapie MeSH
• lidé MeSH
• mnohočetný myelom imunologie   terapie   MeSH
• mucin 1 imunologie   MeSH
• protinádorové vakcíny * MeSH
• T-lymfocyty imunologie   MeSH
• telomerasa imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• epitopy MeSH
• mucin 1 MeSH
• protinádorové vakcíny * MeSH
• telomerasa MeSH
• TERT protein, human MeSH Prohlížeč

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.

• MeSH
• antigen CD83 MeSH
• antigeny CD11c metabolismus   MeSH
• autologní transplantace MeSH
• časové faktory MeSH
• CD antigeny metabolismus   MeSH
• dendritické buňky klasifikace   cytologie   MeSH
• dexamethason aplikace a dávkování   MeSH
• doxorubicin aplikace a dávkování   MeSH
• faktor stimulující kolonie granulocytů aplikace a dávkování   MeSH
• HLA-DR antigeny metabolismus   MeSH
• imunoglobuliny metabolismus   MeSH
• indukce remise MeSH
• kombinovaná terapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• membránové glykoproteiny metabolismus   MeSH
• mnohočetný myelom imunologie   metabolismus   terapie   MeSH
• počet buněk MeSH
• protokoly antitumorózní kombinované chemoterapie terapeutické užití   MeSH
• průtoková cytometrie MeSH
• receptor interleukinu-3 - alfa-podjednotka metabolismus   MeSH
• transplantace periferních kmenových buněk * MeSH
• vinkristin aplikace a dávkování   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD11c MeSH
• CD antigeny MeSH
• dexamethason MeSH
• doxorubicin MeSH
• faktor stimulující kolonie granulocytů MeSH
• HLA-DR antigeny MeSH
• imunoglobuliny MeSH
• membránové glykoproteiny MeSH
• receptor interleukinu-3 - alfa-podjednotka MeSH
• vinkristin MeSH

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.

• MeSH
• buněčné kultury metody   MeSH
• dendritické buňky cytologie   imunologie   MeSH
• imunofenotypizace MeSH
• imunoterapie metody   MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• test smíšené lymfocytární kultury MeSH
• vakcíny imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• vakcíny MeSH

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.

• MeSH
• aktivace lymfocytů MeSH
• biologické markery MeSH
• chromozomální aberace * MeSH
• cytogenetické vyšetření MeSH
• dítě MeSH
• dospělí MeSH
• kultivované buňky MeSH
• lidé MeSH
• lymfocyty chemie   účinky léků   imunologie   MeSH
• mladiství MeSH
• monitorování životního prostředí metody   MeSH
• předškolní dítě MeSH
• těžké kovy analýza   MeSH
• vystavení vlivu životního prostředí škodlivé účinky   analýza   MeSH
• životní styl * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• biologické markery MeSH
• těžké kovy MeSH

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.

• MeSH
• chromozomální aberace * MeSH
• dítě MeSH
• DNA účinky léků   genetika   MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• lymfocyty ultrastruktura   MeSH
• mladiství MeSH
• mutageny škodlivé účinky   MeSH
• novorozenec MeSH
• poškození DNA MeSH
• pracovní expozice MeSH
• předškolní dítě MeSH
• průmysl MeSH
• věkové faktory MeSH
• vystavení vlivu životního prostředí MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA MeSH
• mutageny MeSH