Impact of genomic risk factors on survival after haematopoietic stem cell transplantation for patients with acute leukaemia.

The EBMT risk score is an established tool successfully used in the prognosis of survival post-HSCT and is applicable for a range of haematological disorders. One of its main advantages is that score generation involves summation of clinical parameters that are available pretransplant. However, the EBMT risk score is recognized as not being optimal. Previous analyses, involving patients with various diagnoses, have shown that non-HLA gene polymorphisms influence outcome after allogeneic HSCT. This study is novel as it focuses only on patients having acute leukaemia (N = 458) and attempts to demonstrate how non-HLA gene polymorphisms can be added to the EBMT risk score in a Cox regression model to improve prognostic ability for overall survival. The results of the study found that three genetic factors improved EBMT risk score. The presence of MAL (rs8177374) allele T in the patient, absence of glucocorticoid receptor haplotype (consisting of rs6198, rs33389 and rs33388) ACT in the patient and absence of heat-shock protein 70-hom (+2437) (rs2227956) allele C in the patient were associated with decreased survival time. When compared to the EBMT risk score, the scores combining EBMT risk score with the genetic factors had an improved correlation with clinical outcome and better separation of risk groups. A bootstrapping technique, involving repeated testing of a model using multiple validation sets, also revealed that the newly proposed model had improved predictive value when compared to the EBMT risk score alone. Results support the view that non-HLA polymorphisms could be useful for pretransplant clinical assessment and provide evidence that polymorphisms in the recipient genotype may influence incoming donor cells, suppressing the initiation of the graft versus leukaemia effect and reducing survival.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Predicting survival using clinical risk scores and non-HLA immunogenetics.

Previous studies of non-histocompatibility leukocyte antigen (HLA) gene single-nucleotide polymorphisms (SNPs) on subgroups of patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) revealed an association with transplant outcome. This study further evaluated the association of non-HLA polymorphisms with overall survival in a cohort of 762 HSCT patients using data on 26 polymorphisms in 16 non-HLA genes. When viewed in addition to an already established clinical risk score (EBMT-score), three polymorphisms: rs8177374 in the gene for MyD88-adapter-like (MAL; P=0.026), rs9340799 in the oestrogen receptor gene (ESR; P=0.003) and rs1800795 in interleukin-6 (IL-6; P=0.007) were found to be associated with reduced overall survival, whereas the haplo-genotype (ACC/ACC) in IL-10 was protective (P=0.02). The addition of these non-HLA polymorphisms in a Cox regression model alongside the EBMT-score improved discrimination between risk groups and increased the level of prediction compared with the EBMT-score alone (gain in prediction capability for EBMT-genetic-score 10.8%). Results also demonstrated how changes in clinical practice through time have altered the effects of non-HLA analysis. The study illustrates the significance of non-HLA genotyping prior to HSCT and the importance of further investigation into non-HLA gene polymorphisms in risk prediction.

The scoliosis (sco) mouse: a new allele of Pax1.

We describe the spontaneous mutant mouse scoliosis (sco) that carries a new allele of Pax1 (un-i, undulated intermediate). The Pax1(un-i) allele is lacking the 5'-flanking region and exon 1 to 4 which is mapped to nt -2636 to -640 and -272 to 4271 of the Pax1 gene. Homozygous mice show a mild form of the known phenotypes of other Pax1 mutants. Adult mice have a lumbar scoliosis and kinky tails. In homozygous embryos the skeleton ossifies early, ossification centers of the vertebral bodies are fused with the ossification centers of the pedicles. Neural arches and spinous processes are underdeveloped but the pedicles and transverse processes are overdeveloped which is in contrast to other Pax1 mutants. In the scapula, the acromion is missing and the deltoid tuberosity of the proximal humerus is shortened and thickened. Among the inner organs the thymus development is affected. In late embryos, the thymus is small and thymocyte numbers are reduced. T-cell development from CD4- and CD8- double negative (DN) to CD4+ and CD8+ double positive (DP) is decelerated. The percentage of CD90+ cells is also reduced but in contrast to other Pax1 mutants no alteration of the expression level of the CD90 (Thy-1) could be found.

Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.

Transforming growth factor (TGF)-beta and insulin display opposite effects in regulating programmed cell death during vertebrate retina development; the former induces apoptosis while the latter prevents it. In the present study we investigated coordinated actions of TGF-beta and insulin in an organotypic culture system of early postnatal mouse retina. Addition of exogenous TGF-beta resulted in a significant increase in cell death whereas exogenous insulin attenuated apoptosis and was capable of blocking TGF-beta-induced apoptosis. This effect appeared to be modulated via insulin-induced transcriptional down-regulation of TGF-beta receptor II levels. The analysis of downstream signalling molecules also revealed opposite effects of both factors; insulin provided survival signalling by increasing the level of anti-apoptotic Bcl-2 protein expression and phosphorylation and down-regulating caspase 3 activity whereas pro-apoptotic TGF-beta signalling reduced Bcl-2 mRNA levels and Bcl-2 phosphorylation and induced the expression of TGF-induced immediate-early gene (TIEG), a Krüppel-like zinc-finger transcription factor, mimicking TGF-beta activity.

Physical map and expression profile of genes of the telomeric class I gene region of the rat MHC.

The rat is an important model for studying organ graft rejection and susceptibility to certain complex diseases. The MHC, the RT1 complex, plays a decisive role in controlling these traits. We have cloned the telomeric class I region of the RT1 complex, RT1-C/E/M, of the BN inbred rat strain in a contig of overlapping P1-derived artificial chromosome clones encompassing approximately 2 Mb, and present a physical map of this MHC region. Forty-five class I exon 4-hybridizing BAM:HI fragments were detected, including the previously known rat class I genes RT1-E, RT-BM1, RT1-N, RT1-M2, RT1-M3, and RT1-M4. Twenty-six non-class I genes known to map to the corresponding part of the human and mouse MHC were tested and could be fine mapped in the RT1-C/E/M region at orthologous position. Four previously known microsatellite markers were fine mapped in the RT1-C/E/M region and found to occur in multiple copies. In addition, a new, single-copy polymorphic microsatellite has been defined. The expression profiles of several class I genes and the 26 non-class I genes were determined in 13 different tissues and exhibited restricted patterns in most cases. The data provide further molecular information on the MHC for analyzing disease susceptibility and underline the usefulness of the rat model.

Genomic and funtional aspects of the rat MHC, the RT1 complex.

The major histocompatibility complex (MHC) plays a central role in controlling immune responsiveness, susceptibility to certain diseases and histo-incompatibility. A physical map of the complete rat MHC, the RTI complex, is presented based on a PAC clonal contig (RT1n haplotype). Expression profiling of various tissues of different inbred strains has been carried out for genes of the RT1-C/E/M region, and different types of variability of expression are shown. As an example of single gene analysis, the RT1-linked heat shock-inducible heat shock genes Hsp70-1 and Hsp70-2 have been studied. It is demonstrated that their gene products are able to increase lysability of target cells by cytotoxic T lymphocytes.

Heat shock protein 70 is able to prevent heat shock-induced resistance of target cells to CTL.

Heat shock or transfection with heat shock protein 70 (Hsp70) genes has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity. We have reported previously that heat shock confers resistance to CTL in the rat myeloma cell line Y3 that is Hsp70 defective. Evidence is now presented that Hsp70 is able to prevent the induction of the resistant phenotype. In Con A-stimulated lymphocytes and in lymphocyte x Y3 somatic cell hybrid clones a severe, non-Hsp70-inducing heat shock elicits resistance to CTL in contrast to a heat shock that results in Hsp70 expression. Thus, Hsp70 expression appears to be negatively associated with the development of resistance. Furthermore, loading of Y3 cells with recombinant Hsp70 protein before heat shock is able to prevent resistance. Because apoptosis induced in Y3 cells by heat shock is not affected, Hsp70 appears to interfere selectively with the CTL-induced lethal pathway that is found to be calcium but not caspase dependent. It is suggested that after heat shock Hsp70 enhances the CTL-induced apoptotic pathway by chaperoning certain proteins in the target cell that are involved in the execution of cell death. Thus, although shown to confer protection against many cytotoxic mechanisms, Hsp70 does not appear to be generally cytoprotective. This observation could also be of relevance when interpreting the effectiveness of tumor immunity.

Enhanced susceptibility to cytotoxic T lymphocytes without increase of MHC class I antigen expression after conditional overexpression of heat shock protein 70 in target cells.

Antigenic peptides have been found associated with heat shock proteins (HSP) including cytoplasmic HSP70 and heat shock cognate protein 70 as well as the endoplasmic reticulum-resident glucose-regulated protein 94. Recently, HSP70 transfection has been reported to increase MHC class I cell surface expression and antigen presentation on mouse melanoma B16 cells (Wells et al., Int. Immunol. 1998. 10: 609). To analyze the effect of HSP70 on MHC class I cell surface expression and lysability of target cells we transfected a human melanoma cell line with the rat Hsp70-1 gene using the Tet-On system for conditional overexpression of HSP70. Induction of HSP70 did not increase cell surface expression of HLA class I molecules in general or individual HLA-A and B antigens in particular. Nonetheless, induction of HSP70 enhanced susceptibility of these cells to lysis by allospecific CTL. The same effect was observed using an HLA-A2-restricted tyrosinase-specific CTL clone after pulsing the tyrosinase-negative target cells with the specific peptide. Thus, HSP70 induction can increase killing by CTL without affecting MHC class I cell surface expression or antigen processing. This effect of HSP70 appears to be different from the commonly found protection exerted by HSP70 against stress like heat shock, and might be mediated by improving CTL-induced apoptosis.

Presenilin 2 deficiency causes a mild pulmonary phenotype and no changes in amyloid precursor protein processing but enhances the embryonic lethal phenotype of presenilin 1 deficiency.

Mutations in the homologous presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of familial Alzheimer's disease. Although PS1 function and dysfunction have been extensively studied, little is known about the function of PS2 in vivo. To delineate the relationships of PS2 and PS1 activities and whether PS2 mutations involve gain or loss of function, we generated PS2 homozygous deficient (-/-) and PS1/PS2 double homozygous deficient mice. In contrast to PS1(-/-) mice, PS2(-/-) mice are viable and fertile and develop only mild pulmonary fibrosis and hemorrhage with age. Absence of PS2 does not detectably alter processing of amyloid precursor protein and has little or no effect on physiologically important apoptotic processes, indicating that Alzheimer's disease-causing mutations in PS2, as in PS1, result in gain of function. Although PS1(+/-) PS2( -/-) mice survive in relatively good health, complete deletion of both PS2 and PS1 genes causes a phenotype closely resembling full Notch-1 deficiency. These results demonstrate in vivo that PS1 and PS2 have partially overlapping functions and that PS1 is essential and PS2 is redundant for normal Notch signaling during mammalian embryological development.

Heat-induced expression of MHC-linked HSP70 genes in lymphocytes varies at the single-cell level.

The expression of MHC-linked heat shock protein 70 (HSP70) genes HSP70-1 and HSP70-2 has been studied in human and rat lymphocytes and Concanavalin A (con A)-induced lymphoblasts by in situ hybridization and flow cytometry. In in vitro experiments transcripts of these genes were observed only after heat shock, and mitogen stimulation per se did not lead to induction. Heat shock-induced expression of HSP70-1 and HSP70-2 mRNA varied at the single-cell level. The fraction of HSP70-positive lymphocytes increased continuously with the severity of heat shock. However, 15-35% of the cells always remained HSP70 transcript negative. After fever induction in vivo similar variation of HSP70-1 and HSP70-2 mRNA expression could also be observed. Analysis of heat shocked lymphocytes by flow cytometry demonstrated that HSP70 induction varied also at the protein level, a fraction of cells always remaining unresponsive. Thus, HSP70 induction does not appear to occur in each cell and to a similar extent when a given cell population is exposed to the same stress.

Reduced susceptibility of electroporated tumor cell lines to killing by cytotoxic lymphocytes.

Electroporation is a widely applied method for gene or protein transfer into cells, and it is also used for electrochemotherapy of cancer. During gene transfection studies, electroporation was found to decrease transiently susceptibility of some tumor cell lines to alloreactive cytotoxic T lymphocytes (CTL) or lymphokine-activated killer (LAK) cells. In each cell line electroporation induced c-fos mRNA. In K562 cells HSP70 mRNA induction also occurred. Expression of Grp78, Bcl-2, CD95/Fas, or major histocompatibility complex class I molecules was not affected by electroporation.

Heterogeneous patterns of constitutive and heat shock induced expression of HLA-linked HSP70-1 and HSP70-2 heat shock genes in human melanoma cell lines.

The heat shock response, which is characterized by the induction of heat shock proteins, is known to affect the ability of tumour cells to cope with potentially adverse conditions such as hypoxia, glucose starvation and cytotoxic immune reactions. To assess the heat shock response of melanoma cells, spontaneous and heat shock induced expression of heat shock proteins was analysed in a panel of 17 human melanoma cell lines. Constitutive expression of HSP27, HSP70, HSC70, HSP90alphabeta and GRP94 proteins was found in all the melanoma cell lines, and HSP70 and HSC70 were also induced by heat shock. The major heat inducible HLA-linked HSP70-1 and HSP70-2 genes were analysed at the mRNA level. Basal expression and inducibility varied between the different melanoma cell lines. In addition, in situ hybridization demonstrated heterogeneous expression of these genes among single cells of a given cell line. In general, each melanoma cell line appears to exhibit an individual type of HSP70 expression that might reflect selection during tumour progression and therapy.

Induction of heat shock protein 70 genes in human lymphocytes during fever therapy.

The induction of heat shock protein 70 (HSP70) genes has been studied in peripheral blood mononuclear cells of individuals undergoing fever therapy because of metastatic malignant melanoma. Induction of HSP70 was assessed at the protein level by metabolic labelling or, for the HLA-linked HSP70-1 and HSP70-2 genes, at the RNA level by in situ hybridization. However, de novo expression of HSP70 could be observed during fever (usually above 39 degrees C) in only about half of the cases. No simple threshold model for inducibility of HSP70 in vivo could be applied. The HSP70-1 gene was induced more easily than HSP70-2. Thus, heat-inducible HSP70 genes, including HLA-linked HSP70 genes, become expressed in human lymphocytes during fever, but not regularly.

["Teachers' Training Course"--evaluation of continuing studies for faculty in the field of ethics in medicine]. / "Teachers' Training Course"--Evaluation eines Fortbildungsangebots für Lehrende auf dem Gebiet der Ethik in der Medizin.

The need for continuing education in the field of ethics in health professions is increasing. A project group of the Academy of Ethics in Medicine, Germany, developed and tested a model for a "Teachers' Training Course" to provide continuing education in this field. Evaluation data of the first two seminars are presented. These data demonstrate that the course matches the demands which participants have requested from such seminars.

A pentanucleotide tandem duplication polymorphism in the 3' untranslated region of the HLA-linked heat-shock protein 70-2 (HSP70-2) gene.

A novel two-allele pentanucleotide tandem duplication polymorphism is described within the 3' untranslated region of the HLA-linked HSP70-2 gene, for which a PstI polymorphism is known. All four haplotype combinations were found.

[Teachers' training course. A project of the Academy of Ethics in Medicine]. / Teachers' Training Course. Ein Projekt der Akademie für Ethik in der Medizin.

Needs for continuing education in the field of teaching ethics in health professions are increasing. A project group of the Academy of Ethics in Medicine, Germany, developed and tested a model for a "Teachers' Training Course", based on special educational aims. This project--a "workshop for teachers"--is described.

[Ethical controversy surrounding RU 486: data and arguments]. / Die ethische Auseinandersetzung um RU 486: Daten und Argumente.

By RU 486 being synthesized (1980) a pharmacological alternative to surgical methods of abortion has been created. Since 1988 Mifepristone is applied for termination of pregnancies under severe control. Major pros and cons in debates about RU 486 in medical publications are analyzed, their relations to central ethical principles being worked out clearly. As consequences of the analysis of arguments the authors formulate four conditions for the use of Mifepristone as method of abortion which should be considered in further research and practice.