Differential microRNA expression between decidual and peripheral blood natural killer cells in early pregnancy.

STUDY QUESTION: Have decidual natural killer (dNK) cells a different microRNA (miRNA or miR) expression pattern compared to NK cells circulating in the peripheral blood (pb) of healthy pregnant women in the first trimester of gestation? SUMMARY ANSWER: dNK cells have a unique miRNA profile, showing exclusive expression of a set of miRNAs and significant up- or down-regulation of most of the miRNAs shared with pbNK cells. WHAT IS KNOWN ALREADY: dNK cells differ from pbNK cells both phenotypically and functionally, and their origin is still debated. Many studies have indicated that miRNAs regulate several important aspects of NK cell biology, such as development, activation and effector functions. STUDY DESIGN, SIZE, DURATION: Decidua basalis and peripheral blood specimens were collected from women (n = 7) undergoing voluntary termination of gestation in the first trimester of pregnancy. dNK and pbNK cells were then highly purified by cell sorting. PARTICIPANTS/MATERIALS, SETTING, METHODS: miRNAs expression was analysed by quantitative RT-PCR (qRT-PCR)-based arrays using RNA purified from freshly isolated and highly purified pbNK and dNK cells. Results from arrays were validated by qRT-PCR assays. The bioinformatics tool ingenuity pathway analysis (IPA) was applied to determine the cellular network targeted by validated miRNAs and the correlated biological functions. MAIN RESULTS AND THE ROLE OF CHANCE: Herein, we identified the most differentially expressed miRNAs in NK cells isolated from peripheral blood and uterine decidua of pregnant women. We found that 36 miRNAs were expressed only in dNK cells and two miRNAs only in pbNK cells. Moreover, 48 miRNAs were commonly expressed by both NK cell preparations although at different levels: 28 were upregulated in dNK cells, while 15 were downregulated compared to pbNK cells. Validation of a selected set (n = 11) of these miRNAs confirmed the differential expression of nine miRNAs: miR-10b and miR-214 expressed only in dNK cells and miR-200a-3p expressed only in pbNK cells; miR-130b-3p, miR-125a-5p, miR-212-3p and miR-454 were upregulated while miR-210-3p and miR-132 were downregulated in dNK cells compared to pbNK cells. IPA network analysis identified a single network connecting all the miRNAs as well as their significant involvement in several classes of functions: 'Organismal injury, Reproductive system disease, Inflammatory disease' and 'Cellular development'. These miRNAs target molecules such as argonaute 2, tumour protein p53, insulin and other genes that belong to the same network and significantly influence cell differentiation and pregnancy. LIMITATIONS, REASONS FOR CAUTION: In the present study, the cellular network and biological functions modulated by miRNAs differentially expressed in dNK and pbNK cells were identified by IPA considering only molecules and relationships that were with confidence 'experimentally observed' in leucocytes. The decidual and pbNK cells that were analysed here are a heterogeneous population and further study will help to disentangle whether there are differences in miRNA production by the different subsets of NK cells. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study describing a different miRNA expression profile in dNK cells compared to matched pbNK cells during the first trimester of pregnancy. Our findings improved the body of knowledge on dNK cell biology and strongly suggest further investigation into the roles of miRNAs that are differentially expressed in human dNK compared to pbNK cells. Our results suggest that specific miRNAs can modulate dNK cell origin and functions, highlighting a potential role of this miRNA signature in human development and diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Istituto Pasteur, Fondazione Cenci Bolognetti, the European NoE EMBIC within FP6 (Contract number LSHN-CT-2004-512040), Istituto Italiano di Tecnologia, and Ministero dell'Istruzione, dell'Università e della Ricerca (Ricerche Universitarie), and from Università Politecnica delle Marche. There are no conflicts of interest to declare.

Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

The ATT strand of AAT.ATT trinucleotide repeats adopts stable hairpin structures induced by minor groove binding ligands.

AAT.ATT is the most abundant and also the most frequently polymorphic class of trinucleotide repeats in the human genome. To characterize its structural properties and conformational changes induced by minor groove ligands, (AAT)(6) and (ATT)(6) oligomers as well as their complexes with DAPI were investigated by electrophoretic mobility and UV thermal stability as well as fluorescence and NMR spectroscopy. The results show that individual (AAT)(6) and (ATT)(6) strands exist principally as monomeric non-hydrogen-bonded structures. Their individual interaction with DAPI induces the formation of base-paired structures with different thermal stabilities by quite spectroscopically distinct binding mechanisms. In the presence of DAPI, (ATT)(6) forms a monomeric hairpin structure stabilized by two ligands located in the minor groove with a strong apparent binding constant of 3.4 x 10(6) M(-)(1). The DAPI-induced (ATT)(6) hairpin is characterized by well-stacked A.T Watson-Crick and T.T wobble base pairs, a high electrophoretic mobility, and a melting temperature of 41 degrees C. Interaction of DAPI with the complementary (AAT)(6) strand favors less stable base-paired structures, and the results are consistent with electrostatic and hydrogen-bond interactions of the ligand with the phosphodiester backbone of (AAT)(6) by minor involvement of DNA bases.

[Acute fever without source in infants and children less than 36 months of age] / Febre aguda sem sinais de localização em crianças menores de 36 meses de idade.

OBJECTIVES: The objective of this article is to review current information about fever without source in children less than 36 months of age and to discuss the practical aspects of diagnosis and management.METHODS: Articles from the specific area were reviewed and presented in a practical form with recommendations from the authors.RESULTS: Febrile children comprise a substantial proportion of ambulatory pediatric visits. The majority of children are less than 3 years old. At first examination, there may be considerable overlap in the clinical appearance of children with fever due to viral illness, occult bacteremia and serious bacterial infection. The authors present the recommendations and protocols cited in the literature to assist physicians in managing infants and children with fever without source.CONCLUSION: Although many approaches to the evaluation and management of the febrile infant exist, no diagnostic protocol or therapeutic scheme is optimal for all patients. Pediatricians may individualize their management of these patients, depending on their experience, or interpretation of the recommendations presented in the literature.

Solution structure of DAPI selectively bound in the minor groove of a DNA T.T mismatch-containing site: NMR and molecular dynamics studies.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer [d(GCGATTCGC)]2, containing a central T.T mismatch, has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics and molecular dynamics computations including relaxation matrix refinement. The results show that the DAPI molecule binds in the minor groove of the central region 5'-ATT-3' of the DNA oligomer, which predominantly adopts a duplex structure with a global right-handed B-like conformation. In the final models of the complex, the DAPI molecule is located nearly isohelical with its NH indole proton oriented towards the DNA helix axis and forming a bifurcated hydrogen bond with the carbonyl O2 groups of a mismatched T5 and the T6 residue of the opposite strand. Mismatched thymines adopt a wobble base pair conformation and are found stacked between the flanking base pairs, inducing only minor local conformational changes in global duplex structure. In addition, no other binding mechanisms were observed, showing that minor groove binding of DAPI to the mismatch-containing site is favoured in comparison with any other previously reported interaction with G.C sequences.

Simultaneous and different binding mechanisms of 4',6-diamidino-2-phenylindole to DNA hexamer (d(CGATCG))2. A 1H NMR study.

The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer (d(CGATCG))2 at a 2:1 drug/duplex ratio has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics, and molecular dynamics computations. Intermolecular nuclear Overhauser effects (NOEs), DNA structure perturbations, and resonance shifts induced by binding provide evidence that DAPI interacts with DNA hexamer by two different binding mechanisms, in fast exchange on the NMR time scale, without any significant distortion of the B-type conformation of DNA hexamer. The results indicate that the ligand binds into the minor groove of the central 5'-ATC-3' region of the hexamer and on the outside of the oligomer by a pi,pi-stacking interaction with the terminal C1:G6 base pairs. A model for both binding mechanisms that accounts for all experimental data was generated by molecular mechanics and dynamics calculations based on experimental NOEs. In the minor groove binding, N2 amino group of G2 precludes a deep insertion of phenyl ring of DAPI into the groove. Position and orientation of the drug in the external stacking interaction resemble those suggested for intercalation of DAPI between C:G base pairs.

[Management of a case of maple syrup urine disease--the use of gluco-insulinotherapy]. / Uma experiência terapêutica no manejo da doença da urina do xarope do bordo.

We report here the treatment and poor outcome of a case of Maple Syrup Urine Disease with late diagnosis and retrieval (2 and 5 months, respectively). As the proband had quite high levels of plasmatic leucine (1956 micromol/L for a normal upper limit of 77), we started immediately with a gluco-insulin therapy to produce anabolism in the infant. When leucine has fallen to 275.3 micromol/L, we instituted feeding with branched chain amino acid-free protein and high energy from carbohydrates. After reviewing briefly the clinical, biochemical and therapeutic aspects of this disorder, we comment on the great difficulties of making early diagnosis and of obtaining the specific dietetic formulas to Maple Syrup Urine Disease, in Brazil.

Evidence for DAPI intercalation in CG sites of DNA oligomer [d(CGACGTCG)]2: a 1H NMR study.

The interaction between 4',6-diamidino-2-phenylindole (DAPI) and the DNA oligomer [d(CGACGTCG)]2 has been investigated by proton one- and two-dimensional NMR spectroscopy in solution. Compared with the minor groove binding of the drug to [d(GCGATCGC)]2, previously studied by NMR spectroscopy, the interaction of DAPI with [d(CGACGTCG)]2 appears markedly different and gives results typical of a binding mechanism by intercalation. C:G imino proton signals of the [d(CGACGTCG)]2 oligomer as well as DAPI resonances appear strongly upfield shifted and sequential dipolar connectivities between cytosine and guanine residues show a clear decrease upon binding. Moreover, protons lying in both the minor and major grooves of the DNA double helix appear involved in the interaction, as evidenced principally by intermolecular drug-DNA NOEs. In particular, the results indicate the existence of two stereochemically non-equivalent intercalation binding sites located in the central and terminal adjacent C:G base pairs of the palindromic DNA sequence. Different lifetimes of the complexes were also observed for the two sites of binding. Moreover, due to the fast exchange on the NMR timescale between free and bound species, different interactions in dynamic equilibrium with the observed intercalative bindings were not excluded.

[Short-term survival after heart transplantation: the relationships between preoperative hemodynamics, organ function and postoperative clinical events]. / Sopravvivenza a breve termine dopo trapianto cardiaco: relazioni tra emodinamica preoperatoria, funzione d'organo, ed eventi clinici postoperatori.

AIM OF THE STUDY: Pulmonary hypertension is known to affect prognosis of cardiac allograft recipients. Aim of this study is to elucidate the mechanisms relating preoperative hemodynamics to early post-transplant mortality. METHODS: Hemodynamic and pre- and postoperative clinical data of 122 heart transplant recipients were reviewed with respect to early mortality (within 1 month or in-hospital). The relationships between hemodynamics and mortality were studied by means of univariate and multivariate analysis of absolute data and at different cut-off values of hemodynamic parameters. RESULTS: The following hemodynamic parameters were significantly different between survivors (n = 107) and non-survivors (n = 15): right atrial pressure (7.7 +/- 4.7 vs. 12.1 +/- 8.6 mm Hg, p < 0.004), pulmonary vascular resistance (2.57 +/- 1.44 vs. 3.72 +/- 1.88 Wood units, p < 0.007), pulmonary vascular resistance index (4.43 +/- 2.53 vs. 6.53 +/- 3.28 Wood units x m2, p < 0.005), and transpulmonary gradient (8.8 +/- 4.8 vs. 12.3 +/- 6.4 mm Hg, p < 0.02). Right atrial pressure and pulmonary vascular resistance index showed an independent value at stepwise multiple logistic regression analysis (p < 0.008 and < 0.03 respectively). When mortality was tested using cut-off values, it was significantly higher with right atrial pressure > or = 12 (7/28 vs 8/94, p < 0.05), pulmonary vascular resistance index > or = 8 (6/13 vs 9/109, p < 0.0005), and transpulmonary gradient > or = 15 (5/13 vs 10/109, p < 0.01). High right atrial pressure, pulmonary vascular resistance index, and transpulmonary gradient were associated with higher preoperative bilirubin (p < 0.03), which was significantly superior in non-survivors (1.44 +/- 1.53 vs. 0.83 +/- 0.61 mg/dl, p < 0.02). Postoperatively, severe right ventricular failure, severe renal failure and infections within 1 month were all strongly associated with an increased mortality (p < 0.00003); they were more common in patients with high preoperative right filling pressure (9% vs. 43%, p < 0.00002) and/or high pulmonary vascular resistance index (14% vs. 38%, p < 0.03), in those with high right atrial pressure (9% vs. 35%, p < 0.0009), and in those with high pulmonary vascular resistance index (17% vs. 58%, p < 0.002) respectively. Mortality after acute rejection within 1 month was significantly higher in patients with high preoperative right atrial pressure (8% vs. 57%, p < 0.006). CONCLUSIONS: Besides pulmonary hypertension, elevated preoperative right filling pressure appears to indicate an increased risk of early death after transplantation; pre- and postoperative end-organ dysfunction and post-transplant complications are more common or more threatening in this setting.

1H NMR study of [d(GCGATCGC)]2 and its interaction with minor groove binding 4',6-diamidino-2-phenylindole.

Two-dimensional NMR spectroscopy has been applied to study the solution binding of 4',6-diamidino-2-phenylindole (DAPI) to synthetic DNA duplex [d(GCGATCGC)]2. The structure of the complex at a molar ratio of 1:1 drug:duplex has been investigated. NMR results indicate that DAPI binds selectively in the minor groove of the DNA region containing only two A:T base pairs. The results disagree with conclusions drawn from footprinting experiments and show that the presence of the G3NH2 group in the minor groove does not prevent the binding. A molecular model is proposed that closely resembles the crystal structure previously published for the interaction of DAPI with the dodecamer [d(CGCGAATTCGCG)]2, containing four A:T base pairs in the binding site. In this model, DAPI lies in the minor groove, nearly isohelical, with its aromatic rings adjacent to H4' protons of T5 and C6 deoxyribose and the NH indole group oriented toward the DNA axis. The binding does not perturb the B-type conformation of the duplex, and the DNA oligomer conserves its 2-fold symmetry, indicating that fast exchange dynamics exist between the two stereochemically equivalent binding sites of the palindromic sequence. The binding constant and the exchange rate between free and bound species were also measured by NMR spectroscopy.

Anthracycline gels. II. Spectroscopic study on doxorubicin-lecithin association products.

Thixotropic and thermally reversible gels have been prepared from doxorubicin-lecithin association products (DL12) by addition of salts to their aqueous solutions. The gel formation and the melting profiles have been followed by several spectroscopic techniques (1H NMR, UV-Vis, Circular Dichroism). The transition temperatures increase as the concentration of both the salt and the DL12 is increased, suggesting a progressive closer approach of the gel forming species. The process of the gel formation is cooperative and causes immobilization of the doxorubicin molecules of DL12.

Anthracycline gels. Preparation and some physico-chemical properties.

Gels have been prepared from aqueous solutions of anthracyclines by addition of salts. The gels are thixotropic and thermally reversible. They are stable for several months in the refrigerator and for long times even at room temperature. The gel-solution transition (melting) temperature depends on the concentration of the anthracycline and on the concentration and nature of the added salt. The melting has been followed by 1H-NMR. Only weak intermolecular interactions (stacking and hydrogen bonds) originate the drug network, within which the solvent is entrapped. 1H-NMR and polarimetric data suggest a stacked helical arrangement of the anthracycline molecules. The gelation process is cooperative.

Antitumor effect and cardiotoxicity of a doxorubicin-lecithin association.

A doxorubicin-lecithin preparation was tested in vitro on B16 melanoma and Lewis lung carcinoma cells, and in vivo on C57BL/6 mice inoculated with 3LL cells. Results obtained demonstrated that the preparation possesses the same antitumor activity as doxorubicin. The cardiotoxicity of doxorubicin and of the doxorubicin-lecithin association were studied for 120 days after the end of the treatments in Wistar rats inoculated once a week for 5 weeks with equivalent doses of the drugs. Myocardial lesions were observed in both the groups of animals, but their severity and extent were reduced in rats treated with the doxorubicin-lecithin association, and their onset was also delayed.

Nucleotide pool and the proliferative process: an NMR study of the liver regeneration.

The metabolic changes occurring during liver regeneration after partial hepatectomy have been followed by examination of the perchloric extracts by means of NMR spectroscopy. Proton spectra show an abrupt decrease of glycogen, glucose and nucleotides, which are essentially ribonucleotides, in the first hours after hepatectomy. Nucleotides begin to store up again in the third day after hepatectomy, while glucose and glycogen storage builds up from the second day. 31P data evidence a sharp drop of phosphomonoesters, i.e. monophosphosugars (including AMP) and phosphocholine, and phosphodiesters, i.e. GPC and GPE, soon after hepatectomy and a recovery of the initial levels approximately at the 60th hour and a further increase of PDE at later times. The NMR findings are in agreement with the biochemical knowledge of the course of liver regeneration.

A simple and economical method for studying protein phosphorylation in vivo in the rat brain.

A simple and economical procedure, capable of routine application, is described for the labelling of cerebral phosphoproteins in vivo. [32P]Orthophosphate, in high concentration, was infused into selected brain areas of anaesthetised rats under stereotaxic control. The animals were frozen with liquid N2 and the labelled tissue punched out of frozen thick sections. [32P]Polypeptides were analysed by high-resolution two-dimensional gel electrophoresis. Several phosphoproteins on the gels were provisionally identified, including synapsin I, MAP-2 and an 82-87 kdalton substrate of protein kinase 'C'.

Episodic excitation and changes in aggressive behavior induced by apomorphine in rats subjected to REM sleep deprivation.

Behavioral changes induced by apomorphine in normal, pseudodeprived (control) and REM sleep-deprived AM-2/TOR inbred rats were investigated. Deprived rats exhibited aggressive behavior for nearly 30 min in the absence of administration of drug, this effect not being observed in normal or control rats. The administration of apomorphine (1, 2 and 5 mg/kg) 5 min before the test elicited short periods of aggressive behavior in normal and control rats, but decreased the total duration of aggressive behavior in deprived rats. However, the deprived rats exhibited a more intense aggressive behavior, since the frequency of real fighting events was enhanced. The administration of apomorphine to deprived rats elicited stereotyped behavior. Enhancement of stereotyped behavior by increasing the dose was correlated with a reduction in the duration of aggressive behavior. Apomorphine also induced short episodes of intense excitability, manifested by increased locomotor activity, jumping and vocalization. This behavioral response was termed "episodic excitation". Deprived rats were significantly more sensitive to apomorphine-induced episodic excitation than normal and control rats. The episodic excitation, stereotyped and aggressive behavior displayed by deprived rats, injected with apomorphine, alternated with time. The results demonstrate increased responsiveness to apomorphine after deprivation of REM sleep. The possible mechanism for such interaction is discussed.

Interactions of the histone octamer with single-stranded DNA. Sedimentation analysis and low-angle X-ray diffraction.

A complex between 140-160 nucleotide single-stranded DNA and the octamer of histones was formed and analyzed by electron microscopy and X-ray low angle diffraction. The morphology of the complex is very similar to that of the nucleosome; the diffraction pattern appears less defined than for chromatin showing broader maxima in the same positions. These results strongly suggest that this particle has a geometry very similar to that of the fundamental subunit of chromatin. The possibility of artifacts due to renaturation reaction promoted by histones is ruled out by the analysis of the complex with S1 nuclease and by the formation of a 'nucleosome like' particle using poly(dT) instead of DNA. Association of the histone octamer with either the 140-160 nucleotide single-stranded DNA or the 140-160 bp double-stranded DNA was evaluated at different histone/DNA input ratios. In both cases, the formation of the complex appears to be regulated by comparable association constants, and in both cases the trend of the complexation reaction in function of the temperature is almost the same. These results suggest that an alternative binding of the histone octamer to double-stranded or to single-stranded DNA requires low energy charge and may be involved in the processes of replication and transcription of the 'active chromatin'.

The mode of action of local anesthetics on the calcium pump of brain.

Subcellular vesicles present in brain microsomal fraction take up calcium by an ATP-dependent process which is probably one of the mechanisms involved in the regulation of free calcium ions concentratioon in the cytosol of nerve cells. The experiments described in this paper were designed to test the effect of local anesthetics on this transport system since it is known that cytoplasmic calcium concentration interferes with nerve excitability and conduction and transmitter release. It was found that tetracaine increases the rate of calcium uptake in the range of 0.5 to 3 mM and inhibits calcium uptake in the range of 4 to 7 mM. Lidocaine and procaine increase calcium uptake in the range of 5 to 30 mM and inhibit calcium uptake in the range of 40 to 70 mM. The effects of local anesthetics were also tested on th ATP hydrolysis coupled with calcium uptake and on the ATP in equilibrium Pi exchange which represents the reverse reaction of this transport system. It was found that three local anesthetics inhibit ATP in equilibrium Pi exchange in concentrations which increase calcium uptake and inhibit ATP hydrolysis in concentrations which inhibit calcium uptake. These findings indicate that the enhancement of calcium uptake by the lower concentrations of local anesthetics is due to a decrease of the reverse reaction, whereas inhibition of calcium uptake by the higher concentrations of local anesthetics is due to the blockage of the transport adenosine triphosphatase.