DC Cultures Research Articles

Dendritic Cells Vaccination in BCR/ABL-Positive Chronic Myeloid Leukemia - Final Results of a Phase I/II Study.

T cells recognizing truly leukemia-specific antigens like bcr/abl or other leukemia-associated antigens such as proteinase3 and WT1 have been demonstrated by several groups. In chronic myeloid leukemia (CML), DC and leukemic cells share a common progeny, leading to constitutive expression of putative tumor antigens such as bcr/abl in DC.Aim of our clinical GMP study was the use of autologous DC as a vaccine in patients with chronic phase bcr/abl+ CML, who had not achieved a major cytogenetic response after treatment with imatinib or a-interferon. Primary endpoint of the study were feasibility and toxicity of DC vaccination, secondary endpoints were reduction of bcr/abl+ mononuclear cells in peripheral blood (measured by FISH and quantitative PCR) and induction of an immune response against leukemia-associated antigens. 10 patients were enrolled, 9 patients did receive the vaccine. DC were generated by leukapheresis from peripheral blood monocytes. DC were cultured in RPMI 1640 supplemented with clinical grade FCS in the presence of GM-CSF (100 ng/ml) and IL-4 (1000U/ml). Final maturation was achieved by addition of TNF-a (50 ng/ml) for 3 days. Flowcytometric analysis on day 8/9 of the cell culture showed that DC had a mature phenotype with strong expression of CD80, CD86, CD83 and HLA-DR. In all patients sufficient numbers of functional DC could be generated. By FISH analysis it could be demonstrated that the mean value of bcr/abl-positive cells in the DC cultures was 33% (range: 3–70%). DC were pulsed with KLH for 3–4 hours prior to clinical use. Vaccination was performed by subcutaneous injection in the inguinal region on days 1, 2, 8 and 21 using increasing numbers of DC per injection (1 to 50 x 10e6 cells per injection). 36 vaccinations were performed in nine patients. The follow-up period was three months.There was no severe toxicity, all vaccinated patients developed a strong DTH reaction, mostly after the third vaccination. In 5/9 patients bcr/abl+ PBMC decreased over the vaccination period, in 2/4 patients quantitative PCR also showed a decrease of bcr/abl in peripheral blood. Only in one of these patients this decrease could clearly be attributed to changes in concomitant therapy during the post-vaccination period. In two patients low frequency T cells recognizing the bcr/abl fusion region were detected by ELISpot or tetramer staining. In all patients PBMC showed an increase in the proliferative response against autologous DC over the post-vaccination period, this increase was partially due to immunization against FCS.We conclude that vaccination of CML patients after large scale GMP generation of bcr/abl+ DC is feasible and safe. Leukemic DC are able to induce T cell activation. A decrease in tumor cell burden/circulating bcr/abl+ cells in some patients suggests that vaccination with DC might possibly be useful as post remission therapy in CML patients after imatinib.

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 μg/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 μg/ml and 4.5 ± 1.5 μg/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 109 cfu/ml and decreased to 1.5 (± 0.2) × 109 cfu/ml and 0.5 (± 0.3) × 109 cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.

Effects of several sera on prolactin and GH release from rat pituitary cells in vitro.

AbstractTo study the acute effects of serum and albumin on prolactin (PRL) and growth hormone (GH) release from pituitary cells, 10, 25, and 50% of normal rat (NRS), horse (HS), fetal calf (FCS), or normal human serum (NHS) or 1, 2.5, and 5% of bovine serum albumin (BSA) was administered to dispersed rat pituitary cells (DC) and to clonal rat pituitary mammotrophs (2B8). Further, to obtain information on the nature of the serum factors influencing pituitary hormone release TRH or serum from hypophysectomized rats or heated, or dialyzed rat serum was also added into the culture medium containing DC, or 2B8 cells. Twenty-five and fifty percent of NRS, HS, and NHS consistently stimulated PRL and GH release from DC cultures. In contrast, FCS and BSA had no effect on the release of either hormone. 2B8 cells showed significant increases in the release of PRL during incubation with NRS, HS, NHS, or FCS at all of the concentrations of serum employed. However, the 2B8 cells showed no change in PRL release when BSA...

In vivo and in vitro methylcholanthrene carcinogenesis in diffusion chambers.

Two-compartment diffusion chambers containing murine fibroblasts and a 5% (150 μg) or 1% (30 μg) MCA-discs were placed either in the peritoneal cavity of syngeneic adult mice or incubated in vitro. After 4–28 weeks of culture, groups of 5–6 DC's were recovered at 4 weeks intervals, the MCA discarded, and the contents individually implanted into immunodepressed syngeneic mice. The carcinogen displayed a strong cytotoxicity in vitro but not in vivo with both doses. On the whole, when 5% MCA was applied sarcomas appeared in 22/23 mice implanted with cells of the in vivo DC cultures and in 11/16 mice which received the in vitro cultured cells; with the lower dose, the in vivo induced tumors were 19/22 and the in vitro ones 6/12. In all groups the tumor outgrowth into the assay animals was inversely correlated with the time of exposure to MCA during the culture period.