Banking placental tissue: an optimized collection procedure for genome-wide analysis of nucleic acids.

INTRODUCTION: Banking of high-quality placental tissue specimens will enable biomarker discovery and molecular studies on diseases involving placental dysfunction. Systematic studies aimed at developing feasible standardized methodology for placental collection in a typical clinical setting are lacking. METHODS: To determine the acceptable timeframe for placental collection, we collected multiple samples from first and third trimester placentas at serial timepoints in a 2-h window after delivery, simultaneously comparing the traditional snap-freeze technique to commercial solutions designed to preserve RNA (RNAlater™), and DNA (DNAgard(®)). The performance of RNAlater for preserving DNA was also tested. Nucleic acid quality was assessed by determining the RNA integrity number (RIN) and genome-wide microarray profiling for gene expression and DNA methylation. RESULTS: We found that samples collected in RNAlater had higher and more consistent RINs compared to snap-frozen tissue. Similar RINs were obtained for tissue collected in RNAlater as large (1 cm(3)) and small (∼0.1 cm(3)) pieces. RNAlater appeared to better stabilize the time zero gene expression profile compared to snap-freezing for first trimester placenta. DNA methylation profiles remained quite stable over a 2 h time period after removal of the placenta from the uterus, with DNAgard being superior to other treatments. DISCUSSION AND CONCLUSION: The collection of placental samples in RNAlater and DNAgard is simple, and eliminates the need for liquid nitrogen or a freezer on-site. Moreover, the quality of the nucleic acids and the resulting data from samples collected in these preservation solutions is higher than samples collected using the snap-freeze method and easier to implement in busy clinical environments.

Processing-dependent and -independent pathways for recognition of iodinated contrast media by specific human T cells.

BACKGROUND: One to three percent of patients exposed to intravenously injected iodinated contrast media (CM) develop delayed hypersensitivity reactions. Positive patch test reactions, immunohistological findings, and CM-specific proliferation of T cells in vitro suggest a pathogenetic role for T cells. We have previously demonstrated that CM-specific T cell clones (TCCs) show a broad range of cross-reactivity to different CM. However, the mechanism of specific CM recognition by T cell receptors (TCRs) has not been analysed so far. OBJECTIVE: To determine how T cells specifically recognize CM. METHODS: CM-specific TCCs were generated from human blood of three CM-allergic patients and a specific TCR was transfected into a mouse T cell hybridoma. Functional analysis such as proliferation assays, IL-2 secretion assays, and calcium influx experiments were performed using irradiated, glutaraldehyde-fixed, CM-pre-incubated, human leucocyte antigen (HLA)-DR-matched or -mismatched antigen-presenting cells (APCs), and HLA-blocking antibodies. RESULTS: We identified two mechanisms of T cell stimulation: some TCCs and the transfectant reacted to CM independent of uptake by APCs because proliferation/IL-2 secretion occurred in the presence of glutaraldehyde-fixed APCs, and intracellular calcium increased within seconds after drug addition. Other TCCs required functional APCs, compatible with uptake and presentation of CM on MHC-class II molecules, as implied by three findings: (1) glutaraldehyde fixation of APCs abrogated presentation; (2) CM could not be washed away from CM-pre-incubated APCs; and (3) the optimal pulsing time was 10-20 h. Because allogeneic, MHC-matched, CM-pulsed APCs could induce proliferative responses as well, the ability of CM uptake and presentation is not unique to APCs from patients with CM-induced delayed hypersensitivity. CONCLUSION: Our data suggest that CM may be stimulatory for T cells either by direct binding to the MHC-TCR complex or by binding after uptake and processing by APCs. This questions the assumed inert nature of CM.

Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells.

BACKGROUND: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. METHODS: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta). RESULTS: mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. CONCLUSIONS: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.

Interaction of classical swine fever virus with dendritic cells.

Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte- and in bone marrow-derived DCs. Interestingly, the infected DCs displayed neither modulated MHC nor CD80/86 expression. Stimulation of DCs with IFN-alpha/TNF-alpha or polyinosinic-polycytidylic acid (pIC) induced phenotypic maturation with increased MHC and CD80/86 expression, both with mock-treated and infected DCs. In addition, the T cell stimulatory capacity of CSFV-infected DCs was maintained both in a polyclonal T cell stimulation and in specific antigen-presentation assays, requiring antigen uptake and processing. Interestingly, similar to macrophages, CSFV did not induce IFN-alpha responses in these DCs and even suppressed pIC-induced IFN-alpha induction. Other cytokines including interleukin (IL)-6, IL-10, IL-12 and TNF-alpha were not modulated. Taken together, these results demonstrated that CSFV can replicate in DCs and control IFN type I responses, without interfering with the immune reactivity. These results are interesting considering that DC infection with RNA viruses usually results in DC activation.

Preventing low birth weight: is prenatal care the answer?

OBJECTIVES: To review the evidence of effectiveness of prenatal care for preventing low birth weight (LBW). METHODS: We reviewed original research, systematic reviews, meta-analyses and commentaries for evidence of effectiveness of the three core components of prenatal care--risk assessment, health promotion and medical and psychosocial interventions--for preventing the two constituents of LBW: preterm birth and intrauterine growth restriction (IUGR). RESULTS: Clinical risk assessment will fail to identify the majority of pregnancies at risk for preterm delivery or IUGR. While biophysical and biochemical modalities appear promising, their cost-effectiveness has not been demonstrated, nor can their routine use be recommended in the absence of effective interventions. Smoking cessation programs appear to be modestly effective. There is insufficient evidence to conclude a benefit for nutrition interventions, work counseling or preterm birth education. Only antenatal corticosteroid therapy has demonstrated a clear benefit in the tertiary prevention of preterm delivery. Interventions for which there is insufficient evidence to conclude a benefit include bed rest, hydration, sedation, cerclage, progesterone supplementation, antibiotic treatment, tocolysis without concomitant use of corticosteroids, thyrotropin-releasing hormone, psychosocial support and home visitation. Additionally, there is a paucity of evidence supporting the effectiveness of prenatal interventions, such as low-dose aspirin, bed rest, maternal hyperoxygenation, plasma volume expansion and antenatal fetal assessment, in preventing IUGR or its associated morbidity and mortality. CONCLUSIONS: Neither preterm birth nor IUGR can be effectively prevented by prenatal care in its present form. Preventing LBW will require reconceptualization of prenatal care as part of a longitudinally and contextually integrated strategy to promote optimal development of women's reproductive health not only during pregnancy, but over the life course.