[What is established in cell therapies? : Possibilities and limits in immuno-oncology]. / Was ist gesichert bei den Zelltherapien? : Möglichkeiten und Grenzen in der Immunonkologie.

Cell and gene therapy as part of immuno-oncology has reached an important milestone in medicine. After decades of experience stem cell transplantation is well established with worldwide >1 million transplantations to date. Due to the improved success of the last years using chimeric antigen receptor (CAR) T cells for CD19 positive leukemia and lymphomas, the interest in cellular therapies is continuously increasing. The current review also gives a short overview about donor lymphocytes, antigen-specific T cells, regulatory T cells, natural killer (NK) cells, mesenchymal stromal cells and induced pluripotent stem (iPS) cells in immuno-oncology.

Percentiles of Lymphocyte Subsets in Preterm Infants According to Gestational Age Compared to Children and Adolescents.

Preterm newborns show an increased susceptibility to infections, conceivably related to their immature immune system. To gain further knowledge about the immune development in early preterm infants, we aimed to establish references for lymphocyte subsets and compare the maturation process during hospitalization to healthy term-born children and adolescents. For this purpose, peripheral blood samples (n = 153) were collected from 40 preterm infants, gestational age (GA) 26-30 week between 2nd and 6th day of life, and were monitored in intervals of every 2 or rather 4 weeks until the end of hospitalization. Furthermore, we analysed single sample controls of 10 term neonates. We compared these data with results of a study in healthy children and adolescent (n = 176). Flow cytometry of immune cell subsets was performed as single-platform analysis using 10-colour flow cytometry. Based on preterm's age, our percentile model allows readout of absolute cell count for lymphocytes, B cells, T cells, NK cells, T8 and T4 cells. The median (minimum) value of T-, B- and NK cells after birth was 2800 (600), 790 (120) and 140 (20) cells/µl, respectively. Major differences were found in absolute cell numbers of B cells, and in the frequency of regulatory T cells, most pronounced in the earliest preterm infants (GA 26). Compared to healthy children and adolescents, preterm infants reached lymphocyte counts in between the 5th and 50th percentile when discharging the hospital. This prospective observational study provides reference percentiles for lymphocytes subsets of preterm infants. These data are conducive to interpret immunological capability of preterm infants with possible immune disorders appropriate.

Detection of neuroblastoma cells during clinical follow up: advanced flow cytometry and rt-PCR for tyrosine hydroxylase using both conventional and real-time PCR.

PURPOSE: Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary. METHODS: We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC). RESULTS: Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression. CONCLUSION: TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.

Two different protein-protein interactions in oligomeric complexes of SV40 large T antigen with the cellular oncoprotein p53.

The simian virus 40 (SV40) large T antigen appears in monomers, dimers and various high molecular weight homo-oligomers. EDTA treatment of cell extracts from SV40-infected and -transformed cells leads to a dissociation of the high molecular weight oligomers which can be reconstituted by dialysis against an EDTA free buffer. Hetero-oligomers, composed of T antigen and the oncoprotein p53 become disassembled in the presence of EDTA into forms sedimenting minimally at 7S and maximally at 14S. These low molecular weight T-p53 complexes are resistant to EDTA treatment. Therefore, our results suggest at least two kinds of protein-protein interactions, an EDTA resistant linkage between large T antigen and p53 and an EDTA-sensitive ionic interaction between T antigen molecules in highly oligomeric complexes.

Oligomerization of oncoprotein p53.

Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or transformed cell lines that overexpress p53 allowed us to perform a detailed analysis of the quaternary structure of p53. By means of sucrose density gradient centrifugation, we found in simian virus 40-transformed cells that overexpress p53, in addition to high-molecular-weight T-p53 complexes, low-molecular-weight forms. The level of T-p53 complexes within simian virus 40-transformed cells seemed to be determined by the intracellular concentration of p53. However, the presence of uncomplexed T antigen and p53 indicated that an appropriate modification of at least one of the two proteins appears to be necessary for complex formation. Using different monoclonal antibodies that distinguish between (i) p53 associated with T antigen or heat shock proteins and (ii) p53 in apparently free form, we found p53 from transformed cells always in high-molecular-weight forms. p53 from normal and immortalized cells, however, was found mainly in low-molecular-weight forms. Pulse-labeling experiments revealed that oligomerization of p53 is a very rapid process. Monomeric forms of p53 which could be detected only by 2 min of pulse-labeling were rapidly converted to stable, high-molecular-weight oligomers. Furthermore, our data indicate a correlation between the occurrence of p53 in high-molecular-weight forms and the transformation state of the cell.

Early gene expression and cellular DNA synthesis after stimulation of quiescent NIH3T3 cells with serum or purified simian virus 40.

Like growth factors or hormones, the DNA tumor virus simian virus 40 can stimulate quiescent cells to re-enter the S-phase. Time course experiments revealed similar kinetics for the stimulation of cellular DNA synthesis by growth factors or SV40 infection, although there was a delay in DNA synthesis of about 2 h in the case of the SV40 infection. The analysis of the transcriptional activation of proto-oncogenes in cells stimulated by serum or SV40 infection revealed an enhanced transcription of a common set of proto-oncogenes, including c-myc and c-fos. Early on after infection of quiescent cells SV40 induced in addition the transcription of the c-sis gene which did not occur with UV-irradiated SV40 virus. Furthermore, we could demonstrate the presence of growth stimulating activities in medium from SV40 infected quiescent cells, which triggered quiescent cells to re-enter the cell cycle. These data suggest that SV40 might stimulate quiescent cells by inducing the transcription and ultimately the release of growth-factors.