Microarray Gene Expression Analysis to Evaluate Cell Type Specific Expression of Targets Relevant for Immunotherapy of Hematological Malignancies.

Cellular immunotherapy has proven to be effective in the treatment of hematological cancers by donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation and more recently by targeted therapy with chimeric antigen or T-cell receptor-engineered T cells. However, dependent on the tissue distribution of the antigens that are targeted, anti-tumor responses can be accompanied by undesired side effects. Therefore, detailed tissue distribution analysis is essential to estimate potential efficacy and toxicity of candidate targets for immunotherapy of hematological malignancies. We performed microarray gene expression analysis of hematological malignancies of different origins, healthy hematopoietic cells and various non-hematopoietic cell types from organs that are often targeted in detrimental immune responses after allogeneic stem cell transplantation leading to graft-versus-host disease. Non-hematopoietic cells were also cultured in the presence of IFN-γ to analyze gene expression under inflammatory circumstances. Gene expression was investigated by Illumina HT12.0 microarrays and quality control analysis was performed to confirm the cell-type origin and exclude contamination of non-hematopoietic cell samples with peripheral blood cells. Microarray data were validated by quantitative RT-PCR showing strong correlations between both platforms. Detailed gene expression profiles were generated for various minor histocompatibility antigens and B-cell surface antigens to illustrate the value of the microarray dataset to estimate efficacy and toxicity of candidate targets for immunotherapy. In conclusion, our microarray database provides a relevant platform to analyze and select candidate antigens with hematopoietic (lineage)-restricted expression as potential targets for immunotherapy of hematological cancers.

The impact of obesity on outcomes of midwife-led pregnancy and childbirth in a primary care population: a prospective cohort study.

OBJECTIVE: To assess the impact of obesity on the likelihood of remaining in midwife-led care throughout pregnancy and childbirth. DESIGN: Secondary analysis of data from a prospective cohort study. SETTING: Dutch midwife-led practices. POPULATION: A cohort of 1369 women eligible for midwife-led care after their first antenatal visit. METHODS: First-trimester body mass index (BMI) was calculated as weight measured at booking divided by height squared. Obstetric data were retrieved from medical records. Multiple logistic regressions were performed to examine the effects of BMI classification on midwife-led pregnancies and childbirths. MAIN OUTCOME MEASURES: Percentages of women remaining in midwife-led care throughout pregnancy and throughout childbirth. RESULTS: Of women in obesity classes II and III, 55% remained in midwife-led care throughout pregnancy and 30% remained in midwife-led care throughout birth. Compared with women of normal weight, women in obesity classes II and III had fewer midwife-led pregnancies (OR 0.38, 95% CI 0.21-0.69), and women who were overweight or in obesity class I had fewer midwife-led childbirths (OR 0.63, 95% CI 0.44-0.90; OR 0.49, 95% CI 0.29-0.84, respectively). Compared with women of normal weight, women who were obese had higher referral rates for hypertensive disorders (4 versus 14%), prolonged labour (4.6 versus 10.4%), and intrapartum pain relief (4 versus 10.4%). The women who were eligible for midwife-led birth and who were overweight or obese, had no more urgent referrals than women of normal weight. Women who were obese and who completed a midwife-led birth had no more adverse outcomes than women of normal weight, with the exception of higher rates of large for gestational age (LGA) babies (>97.7 centile; 12.1%, versus 1.9% in normal weight and versus 3.3% in overweight women). CONCLUSIONS: Although fewer women who were obese remain in midwife-led care during pregnancy and childbirth, there was no increased risk of unfavourable birth outcomes for women who were obese and eligible for a midwife-led birth when compared with women of normal weight. This indicates that when primary care midwives use a risk assessment tool throughout pregnancy and childbirth they are able to safely assign women who are obese to either midwife-led or obstetrician-led care.

Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target.

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

A specific lysine in c-Jun is required for transcriptional repression by E1A and is acetylated by p300.

The adenovirus E1A protein regulates transcription of cellular genes via its interaction with the transcriptional coactivators p300/CBP. The collagenase promoter activated by the c-Jun protein is repressed by E1A. Here we show that E1A repression is specific for c-Jun, as E1A does not repress the collagenase promoter activated by the homologous transcription factor EB1. Using chimeras of c-Jun and EB1, we demonstrate that a 12 amino acid region in the basic region of the c-Jun DNA-binding domain is essential for repression by E1A. Since repression requires the binding of p300 to E1A, we studied the involvement of p300 acetyltransferase activity in the repression mechanism. We demonstrate that c-Jun is acetylated in vivo, and mutational analysis identified Lys271 in the c-Jun basic region to be essential for repression of the collagenase promoter by E1A. In addition, Lys271 is acetylated both in vitro and in vivo. These results suggest that the specific repression of the collagenase promoter by E1A involves acetylation of c-Jun.

The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways.

Initiation factor eIF4E binds to the 5'-cap of eukaryotic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through its own phosphorylation and through inhibitory binding proteins (4E-BPs), which modulate its availability for initiation complex assembly. eIF4E phosphorylation is enhanced by phorbol esters. We show, using specific inhibitors, that this involves both the p38 mitogen-activated protein (MAP) kinase and Erk signaling pathways. Cell stresses such as arsenite and anisomycin and the cytokines tumor necrosis factor-alpha and interleukin-1beta also cause increased phosphorylation of eIF4E, which is abolished by the specific p38 MAP kinase inhibitor, SB203580. These changes in eIF4E phosphorylation parallel the activity of the eIF4E kinase, Mnk1. However other stresses such as heat shock, sorbitol, and H2O2, which also stimulate p38 MAP kinase and increase Mnk1 activity, do not increase phosphorylation of eIF4E. The latter stresses increase the binding of eIF4E to 4E-BP1, and we show that this blocks the phosphorylation of eIF4E by Mnk1 in vitro, which may explain the absence of an increase in eIF4E phosphorylation under these conditions.

Translational properties of the untranslated regions of the p10 messenger RNA of Autographa californica multicapsid nucleopolyhedrovirus.

Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.

Heat shock increases the association of binding protein-1 with initiation factor 4E.

The effects of heat shock on the regulation of the cap-binding initiation factor 4E (eIF4E) and its inhibitory binding protein, 4E-BP1, have been examined in Chinese hamster ovary cells and in cardiac myocytes. Heat shock increased the association between eIF4E and 4E-BP1, and this was associated with a dephosphorylation of 4E-BP1. These effects did not appear to be due wholly to decreased activity of the p70 S6 kinase pathway, which is implicated in the control of 4E-BP1, and they were not mediated by the stress-activated p38 microtubule-associated protein kinase pathway. Increased binding of 4E-BP1 to eIF4E correlated with a decrease in the amount of eIF4G which co-purified with the latter. This could account for the previously observed impairment of eIF4F function during heat shock, and, since heat shock protein mRNAs are believed to be relatively cap-independent, could provide a mechanism for the selective up-regulation of the synthesis of heat shock proteins and other stress proteins during heat shock.

L-malate in golden delicious apples as a possible parameter of acceptability.

Several batches of Golden Delicious apples from three geographical regions in the Netherlands were stored at 4 degrees C in a controlled atmosphere (CA) of 9% CO2, 12% 02 and under normal atomspheric conditions (Air = A). They were judged with the aid of a palatability test after which it was established that the internal quality of a population of fruits remained satisfactory until they reached a range of L-malate of 0.39-0.45 g per 100 g initial fresh weight.