An expression signature at diagnosis to estimate prostate cancer patients' overall survival.

BACKGROUND: This study aimed to identify biomarkers for estimating the overall and prostate cancer (PCa)-specific survival in PCa patients at diagnosis. METHODS: To explore the importance of embryonic stem cell (ESC) gene signatures, we identified 641 ESC gene predictors (ESCGPs) using published microarray data sets. ESCGPs were selected in a stepwise manner, and were combined with reported genes. Selected genes were analyzed by multiplex quantitative polymerase chain reaction using prostate fine-needle aspiration samples taken at diagnosis from a Swedish cohort of 189 PCa patients diagnosed between 1986 and 2001. Of these patients, there was overall and PCa-specific survival data available for 97.9%, and 77.9% were primarily treated by hormone therapy only. Univariate and multivariate Cox proportional hazard ratios and Kaplan-Meier plots were used for the survival analysis, and a k-nearest neighbor (kNN) algorithm for estimating overall survival. RESULTS: An expression signature of VGLL3, IGFBP3 and F3 was shown sufficient to categorize the patients into high-, intermediate- and low-risk subtypes. The median overall survival times of the subtypes were 3.23, 4.00 and 9.85 years, respectively. The difference corresponded to hazard ratios of 5.86 (95% confidence interval (CI): 2.91-11.78, P<0.001) for the high-risk subtype and 3.45 (95% CI: 1.79-6.66, P<0.001) for the intermediate-risk compared with the low-risk subtype. The kNN models that included the gene expression signature outperformed the one designed on clinical parameters alone. CONCLUSIONS: The expression signature can potentially be used to estimate overall survival time. When validated in future studies, it could be integrated in the routine clinical diagnostic and prognostic procedure of PCa for an optimal treatment decision based on the estimated survival benefit.

Neuroblastoma cells injected into experimental mature teratoma reveal a tropism for embryonic loose mesenchyme.

Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem­cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell­induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non­random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

Horse embryonic stem cell lines from the proliferation of inner cell mass cells.

Inner cell mass (ICM) cells were isolated immunosurgically from day 7-8 horse blastocysts and, after proliferation in vitro for 15-28 passages, three lines of cells were confirmed to be embryonic stem (ES) cells by their continued expression of alkaline phosphatase activity and their ability to bind antisera specific for the recognized stem cell markers, SSEA-1, TRA-1-60, TRA-1-81, and the key embryonic gene Oct-4. When maintained under feeder cell-free conditions in vitro, the three lines of cells differentiated into cells of ectodermal, endodermal, and mesodermal lineages. However, they did not form teratomata when injected into the testes of severe combined immunodeficiency (SCID)/beige immunoincompetent mice, thereby indicating a significant difference in phenotype between ES cells of the horse and those of the mouse and human.

Viscoelastic and histologic properties in scarred rabbit vocal folds after mesenchymal stem cell injection.

OBJECTIVE/HYPOTHESIS: The aim of this study was to analyze the short-term viscoelastic and histologic properties of scarred rabbit vocal folds after injection of human mesenchymal stem cells (MSC) as well as the degree of MSC survival. Because MSCs are antiinflammatory and regenerate mesenchymal tissues, can MSC injection reduce vocal fold scarring after injury? STUDY DESIGN: Twelve vocal folds from 10 New Zealand rabbits were scarred by a localized resection and injected with human MSC or saline. Eight vocal folds were left as controls. MATERIAL AND METHODS: After 4 weeks, 10 larynges were stained for histology and evaluation of the lamina propria thickness. Collagen type I content was analyzed from six rabbits. MSC survival was analyzed by fluorescent in situ hybridization staining from three rabbits. Viscoelasticity for 10 vocal folds was analyzed in a parallel-plate rheometer. RESULTS: The rheometry on fresh-frozen samples showed decreased dynamic viscosity and lower elastic modulus (P<.01) in the scarred samples injected with MSC as compared with the untreated scarred group. Normal controls had lower dynamic viscosity and elastic modulus as compared with the scarred untreated and treated vocal folds (P<.01). Histologic analysis showed a higher content of collagen type 1 in the scarred samples as compared with the normal vocal folds and with the scarred folds treated with MSC. MSCs remained in all samples analyzed. CONCLUSIONS: The treated scarred vocal folds showed persistent MSC. Injection of scarred rabbit vocal folds with MSC rendered improved viscoelastic parameters and less signs of scarring expressed as collagen content in comparison to the untreated scarred vocal folds.

In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells.

Previous studies in several laboratories have demonstrated inadvertent chromosomal abnormalities in long-term cultured human embryonic stem cells (HESC). Here, using a two-step selection process we report a functional adaptation of a HESC line, HS181, towards a decreased dependence of extra cellular matrix (ECM) for in vitro survival, that is for growth directly onto a plastic surface. Successful adaptation was paralleled with a karyotype change in 100% of the cells to 47,XX,del(7)(q11.2),+i(12)(p10). The resulting adapted population showed increased survival and growth on plastic and also maintained expression of HESC markers, but showed a decreased pluripotency, as demonstrated by results from embryoid body (EB) formation in vitro. The finding of reduced pluripotency may not be totally unexpected since the variant cells were selected for self-renewal and proliferation, not differentiation during the adaptation to growth on plastic. In the light of recent models of a germ cell origin of HESC it is of particular interest that similar to many of the reported spontaneous HESC mutants, one of the identified specific chromosome abnormalities, i(12p), has also been strongly implicated for human germ cell cancer. However, the mutated HESC variant carrying this mutation failed to grow as a xeno-graft in a mouse model in vivo. This is surprising and needs a further mechanistic analysis for its explanation. Increased knowledge of genetic integrity of HESC may have significance on the understanding of mechanisms for tumor progression and thus strategy for treatments, particularly for tumors occurring in early life.

Real-time reverse transcription-polymerase chain reaction analysis of translation initiation factor 1A (eIF-1A) in human and mouse preimplantation embryos.

Fluorescence-monitored real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to study steady state concentrations of translation initiation factor eIF-1A mRNA in mouse and human preimplantation embryos. Its expression in human embryos has not been described previously. Human oocytes, and 2-cell and 4-cell embryos all showed comparable total concentrations of eIF-1A RNA, indicating a gradual decrease in the average concentration per blastomere during these developmental stages. A 4-fold increase was observed in the 8-cell embryos. This concentration remained at the morula stage, followed by a 7- to 8-fold further increase at the blastocyst stage. Mouse preimplantation embryos already showed increased concentrations of eIF-1A RNA at the 2-cell stage. Thus, transcription levels of the eIF-1A gene are associated with embryonic gene activation (EGA) in both species. The method used, real time RT-PCR, proved to be sensitive enough to detect quantitative expression in single mouse blastomeres, the observed values for steady-state concentrations of mRNA in single blastomeres correlating well with the values for whole embryos. The possibility to study gene expression quantitatively in single blastomeres may be useful in preimplantation genetic diagnosis.

Single cell CGH analysis reveals a high degree of mosaicism in human embryos from patients with balanced structural chromosome aberrations.

We have performed comparative genomic hybridization (CGH) analysis of single blastomeres from human preimplantation embryos of patients undergoing preimplantation genetic diagnosis (PGD) for inherited structural chromosome aberrations and from embryos of IVF couples without known chromosomal aberrations. The aim was to verify the PGD results for the specific translocation, reveal the overall genetic balance in each cell and visualize the degree of mosaicism regarding all the chromosomes within the embryo. We successfully analysed 94 blastomeres from 28 human embryos generated from 13 couples. The single cell CGH could verify most of the unbalanced translocations detected by PGD. Some of the embryos exhibited a mosaic pattern regarding the chromosomes involved in the translocation, and different segregation could be seen within an embryo. In addition to the translocations, we found a high degree of numerical aberrations including monosomies, trisomies and duplications or deletions of parts of chromosomes. All of the embryos (100%) were mosaic, containing more than one chromosomally uniform cell line, or even chaotic with a different chromosomal content in each blastomere.

Clinical outcome of treatment cycles using preimplantation genetic diagnosis for structural chromosomal abnormalities.

OBJECTIVES: To explore oocyte recovery, embryo quality, the number of transferable embryos and pregnancy rate after preimplantation genetic diagnosis (PGD) in patients with structural chromosomal aberrations. METHODS: PGD was performed in seven couples with Robertsonian translocations (Rob), eight couples with reciprocal translocations (Rec), two couples with inversions and one couple with a deletion. A total of 43 treatment cycles were carried out. RESULTS: A total of 14.2 oocytes per cycle were retrieved. Fertilisation and cleavage rates were 63% and 58%, respectively. Of the biopsied embryos 20% were transferable. Comparison of the Rob and Rec group revealed no significant differences in number of oocytes, fertilisation or cleavage rates. The number of transferable embryos after biopsy was significantly higher in the Rob group than in the Rec group. When embryo transfer (ET) was performed the pregnancy rate did not differ between the Rob and the Rec groups. Twenty-eight embryo transfers (one or two embryos) were carried out leading to eight clinical pregnancies (29% per ET): two twins, four singletons, one miscarriage and one ectopic pregnancy. All the children are carriers of balanced chromosomal aberrations. CONCLUSION: An acceptable pregnancy rate can be achieved among couples with structural chromosomal abnormalities.

[Ethical aspects of stem cell research. Legislation and guidelines in Europe]. / Etiska aspekter på stamcellsforskning. Lagar och riktlinjer i Europa.

Research on different types of stem cells is of major interest because of its apparent very promising therapeutic prospects, such as for Parkinson's and Alzheimer's disease, spinal cord injuries, stroke, diabetes, cardiac failure, liver failure, cartilage injuries, severe blood diseases, cancer etc. Stem cells can be derived from different sources: adult tissue, foetal tissues, and from in vitro fertilised embryos. Depending on their origin they have varying capacity to multiply and differentiate to other cell types. It is at present not possible to predict which types of cells will be best suitable for various therapeutic situations. Embryonic stem cells have been shown capable of differentiating into all the different tissues and cell types of the body, but they cannot form a new individual. Because of the ethics question involved, The European Group on Ethics on Science and New Technologies for the European Commission and Parliament (EGE), and the Ethics Committee of the Nordic Council of Ministers have prepared reports and given guidelines for research on stem cells. According to the guidelines, every country should regulate the research. Only embryos, which cannot be used in infertility treatment, and have been donated for research, can be used. Creation of embryos solely for research purposes, including somatic cell nuclear transfer, is not regarded as acceptable for the time being. Both partners of the donating couple have to sign an informed consent document. Ongoing research in Sweden is well in line with these European and Nordic recommendations.

Smaller amounts of antiretroviral drugs are needed when combined with an active ribozyme against HIV-1.

We have tested for combined anti-HIV-1 effects of a hammerhead ribozyme and antiretroviral drugs and the possibility of reducing the drug burden of patients on highly active antiretroviral therapy (HAART). The antiretroviral compounds used represent the three groups in HAART: nucleoside analogue reverse-transcriptase inhibitors, nonnucleoside reverse-transcriptase inhibitors, and protease inhibitors. A human T cell line (HUT78), stably expressing a hammerhead ribozyme targeted to nef (hhRz.nef(9016-9029)), was infected with HIV-1(SF2) in the presence of a single drug. The combined effects on HIV-1 replication were measured by p24 antigen determinations over a 2-week period. In the presence of the ribozyme, smaller amounts of antiretroviral drugs were required to reduce the HIV-1 p24 levels equally as much as when only drugs were present. The results support a strategy of combining ribozyme gene therapy with HAART to improve the long-term outcome of anti-HIV-1 therapy.

Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor.

Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.

Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers.

We have developed preimplantation genetic diagnosis (PGD) for carriers of chromosomal abnormalities using fluorescent in situ hybridisation (FISH). Here we present the detailed analysis of 64 biopsied, normally developing embryos obtained from four Robertsonian and three reciprocal translocation carriers in 11 treatment cycles of which four resulted in normal pregnancies (three simplex, one duplex). In order to investigate the degree of mosaicism and segregation mode in the embryos, the primary analysis of the biopsied cells was extended with the analysis of all cells from the non-transferred embryos. The analysis also included a second hybridisation with two additional probes, not involved in the translocation (chromosomes 1 and 9), in order to investigate the overall degree of mosaicism. Seventeen out of 64 analysed embryos were balanced for the chromosomes involved in the translocation and 14 of these were transferred. Forty-seven out of 64 embryos (73%) were mosaic regarding the chromosomes involved in the translocation and alternate segregation mode was the most common mode of segregation. Moreover, we have found a higher degree of mosaicism for the chromosomes involved in translocations as compared to control chromosomes. This difference was more pronounced for the embryos from reciprocal translocation carriers. The results, mechanisms, significance and implications of our findings are discussed.

Dose-response resistance to HIV-1/MuLV pseudotype virus ex vivo in a hairpin ribozyme transgenic mouse model.

We have investigated the efficacy of a hairpin ribozyme targeting the 5' leader sequence of HIV-1 RNA in a transgenic model system. Primary spleen cells derived from transgenic or control mice were infected with HIV-1/MuLV pseudotype virus. A significantly reduced susceptibility to infection in ribozyme-expressing transgenic spleen cells (P = 0.01) was shown. Variation of transgene-expression levels between littermates revealed a dose response between ribozyme expression and viral resistance, with an estimated cut off value below 0.2 copies of hairpin ribozyme per cell. These findings open up possibilities for studies on ribozyme efficacy and anti-HIV-1 gene therapy.

PDGFB regulates the development of the labyrinthine layer of the mouse fetal placenta.

PDGFB is a growth factor which is vital for the completion of normal prenatal development. In this study, we report the phenotypic analysis of placentas from mouse conceptuses that lack a functional PDGFB or PDGFRbeta gene. Placentas of both types of mutant exhibit changes in the labyrinthine layer, including dilated embryonic blood vessels and reduced numbers of both pericytes and trophoblasts. These changes are seen from embryonic day (E) 13.5, which coincides with the upregulation of PDGFB mRNA levels in normal placentas. By E17, modifications in shape, size, and number of the fetal blood vessels in the mutant placentas cause an abnormal ratio of the surface areas between the fetal and the maternal blood vessels in the labyrinthine layer. Our data suggest that PDGFB acts locally to contribute to the development of the labyrinthine layer of the fetal placenta and the formation of a proper nutrient-waste exchange system during fetal development. We point out that the roles of PDGFB/Rbeta signaling in the placenta may be analogous to those in the developing kidney, by controlling pericytes in the labyrinthine layer and mesangial cells in the kidney.

Squamous cell carcinomas and increased apoptosis in skin with inhibited Rel/nuclear factor-kappaB signaling.

The Rel/nuclear factor-kappaB (Rel/NF-kappaB) transcription factors have been implicated previously in control of apoptosis, cell proliferation, and oncogenesis. Here we show that selective inhibition of Rel/NF-kappaB signaling in murine skin, by targeted overexpression of a super-repressor form of IkappaB-alpha, results in an increased basal frequency of apoptotic cells and the spontaneous development of squamous cell carcinomas. Presence of hyperplasia and hair follicle degeneration demonstrate an important role for Rel/NF-kappaB signaling in normal epidermal development and homeostasis. Transgenic skin, in addition, showed an enhanced sensitivity to UV-induced apoptosis. These data suggest an involvement of the Rel/NF-kappaB signaling pathway in apoptosis and cancer development of the skin.

A high degree of aneuploidy in frozen-thawed human preimplantation embryos.

We have studied the chromosomal content in 68 normally fertilised freeze-thawed human embryos of good morphology from 34 patients with an average maternal age of 32,6 years. Forty embryos showed post-thaw cellular division and twenty-eight post-thaw cleavage arrest. After spreading of the embryos on microscope slides, analysis of chromosomes X, Y, 15, 16, 17 and 18 was performed using two rounds of fluorescent in situ hybridisation (FISH). According to the results, the embryos were divided into four groups: (I) normal, all nuclei uniformly diploid, (II) diploid mosaics, normal diploid blastomeres in combination with abnormal blastomeres, (III) abnormal, all nuclei abnormal, (IV) chaotic, the chromosome constitution varies randomly from cell to cell. Approximately 25% of the embryos had normal number of the chromosomes tested, while the majority of the embryos were abnormal. Most of the abnormal embryos were diploid mosaics (57%). This was true for the embryos showing cleavage division as well as the embryos showing cleavage arrest. Our data show a slightly higher incidence of abnormal embryos compared to those obtained with FISH in non-cryopreserved embryos and confirm that the majority of preimplantation embryos fertilised in vitro contain abnormal blastomeres. The results, mechanisms, significance and implications are discussed.

Telomere loss in peripheral blood mononuclear cells may be moderately accelerated during highly active antiretroviral therapy (HAART).

It has been speculated that infection with HIV-1 may lead to a significant increase in turnover, and subsequent exhaustion, of immune repopulation. Given that telomeric DNA is lost on mitotic replication, telomere lengths can be used as an indirect gauge of this rate. We have analyzed the mean telomere restriction fragment lengths in peripheral blood mononuclear cells (PBMC) from 31 patients with established, though mainly untreated, HIV infection and found them to be no different than those among healthy controls. Our results are in line with several findings in CD4+ cell fractions but contradict a previous report suggesting that telomere shortening contributes to immune failure. Interestingly, after approximately 2 years of subsequent aggressive antiretroviral treatment we found a telomere reduction corresponding to a loss of about 250 base pairs per year; this is roughly tenfold above that predicted from healthy individuals. This could partly result from nucleoside analogue inhibition of the natural telomere replacement enzyme, telomerase-a reverse transcriptase inducible in certain hematopoietic cells. However, this may also indicate accelerated cell replacement on initiation of optimal therapeutic regimes or result from changes in the composition of the PBMC pool. These results suggest careful monitoring of telomere lengths during long-term HAART.

Preimplantation genetic diagnosis of DiGeorge syndrome.

We report the first case of preimplantation genetic diagnosis used in order to avoid chromosomal imbalance in the progeny of a woman mildly affected by DiGeorge syndrome and carrier of a microdeletion of chromosome 22q11.2. In total, seven embryos were biopsied in three separate treatments and analysed by fluorescent in-situ hybridization (FISH). Of these, four were carrying the deletion, two were normal and in one the analysis was inconclusive. The diagnostic procedure was performed within 5 h. This allowed the biopsied embryos to be transferred the same day as the biopsy was taken (day 3). Two embryos were transferred in the third treatment, but no pregnancy was established. Patients with a 22q11 microdeletion, who have a 50% risk of transmitting the deletion to their offspring, can now be offered preimplantation genetic diagnosis using FISH for the detection of a 22q11 deletion.

Preimplantation genetic diagnosis of a large pericentric inversion of chromosome 5.

We report the first established pregnancy using preimplantation genetic diagnosis in order to avoid chromosomal imbalance in the progeny of a woman carrying a large inversion of chromosome 5. This is also the first time where it has been possible to study the distribution of balanced and unbalanced gametes in a female inversion carrier. In total, 23 embryos were biopsied in two separate treatments and analysed by fluorescent in-situ hybridization. Of these, 10 were unbalanced, nine were balanced and for four the analysis was inconclusive. The diagnostic procedure was performed within 3.5 h. This allowed the biopsied embryos to be transferred the same day as the biopsy was taken (day 3). Two embryos were transferred each time, and in the second treatment a twin pregnancy with two chromosomally balanced fetuses was established. Healthy twins were delivered at 34 weeks of gestation.

IL-6 antisense oligonucleotides inhibit IgE production in IL-4 and anti-CD40-stimulated human B-lymphocytes.

Stimulation of tonsillar B-lymphocytes with CD40 antibodies and IL-4 leads to homotypic adhesion, proliferation, and differentiation into Ig-producing cells. It also leads to the production of IL-6, a pleiotropic cytokine involved in B-cell maturation and differentiation. To assess the importance of IL-6 in the differentiation process, an antisense oligonucleotide to IL-6 was added to tonsillar B-cells together with CD40 antibodies and IL-4. This led to clearly reduced levels of IL-6 as well as to a specific inhibition of IgE production. Also, IgG secretion was somewhat reduced while IgM appeared to be unaffected. The effects were not due to toxicity of the oligonucleotide since proliferation proceeded normally or was slightly enhanced in the presence of the antisense. The findings show that endogenous IL-6 is an important co-factor for the generation of B-cells secreting IgE and IgG but that it is not required for IgM production. They further indicate that IL-6 may not be necessary as a co-factor in CD40/IL-4 induced proliferation.