External validation of a simple risk score based on the ASPRE trial algorithm for preterm pre-eclampsia considering maternal characteristics in nulliparous pregnant women: a multicentre retrospective cohort study.

OBJECTIVE: To validate the performance of a first-trimester simple risk score based on the ASPRE trial algorithm for pre-eclampsia. DESIGN: Multicentre retrospective cohort analysis. SETTING: Four Italian hospitals. POPULATION: Unselected nulliparous women at 11-13 weeks of gestation from January 2014 through to January 2018. METHODS: Model performance was evaluated based on discrimination and calibration. MAIN OUTCOME MEASURES: Delivery before 37 weeks of gestation with a diagnosis of pre-eclampsia. RESULTS: Based on 73 preterm pre-eclampsia cases and 7546 controls (including 101 cases of late pre-eclampsia), the area under the receiver operating characteristics curve was 0.659 (95% CI 0.579-0.726). The sensitivity was 32.9% (95% CI 22.1-43.7) at a false-positive rate of 8.8%. The positive likelihood ratio was 3.74 (95% CI 2.67-5.23), the positive predictive value was 3.49% (95% CI 2.12-4.86%) and the negative predictive value was 99.3% (95% CI 99.1-99.5%). The sensitivity and positive likelihood ratio were approximately 40% lower than in the original study. The calibration analysis showed a good agreement between observed and expected risks (P = 0.037). Comparison with the Fetal Medicine Foundation (FMF) algorithm yielded a difference in the area under the curve of 0.084 (P = 0.007). CONCLUSIONS: In our Italian population, the simple risk score had a lower performance than expected for the prediction of preterm pre-eclampsia in nulliparous women. The FMF algorithm applied to the same data set resulted in a better prediction. TWEETABLE ABSTRACT: Simple risk score predicts preterm pre-eclampsia in Italy.

Multivariable evaluation of maternal hemodynamic profile in pregnancy complicated by fetal growth restriction: prospective study.

OBJECTIVE: To evaluate the association between fetal growth restriction (FGR) and maternal hemodynamic parameters using multivariable analysis, adjusting for major confounding factors, such as hypertensive disorders of pregnancy (pre-eclampsia and gestational hypertension). METHODS: A prospective cohort study was conducted between January 2013 and April 2016. Two cohorts of patients were recruited, between 24 and 39 weeks of gestation, in a high-risk outpatient setting. These cohorts comprised 49 appropriate-for-gestational-age singleton fetuses and 93 that were FGR (abdominal circumference (AC) at recruitment in the second half of pregnancy ≤ 10th percentile with a previous normal AC at 20-22 weeks). Maternal echocardiography was performed at the time of enrolment and included hemodynamic parameters of systolic and diastolic function and cardiac remodeling indices. Data were analyzed using a multivariable generalized linear model to estimate the association of FGR with maternal hemodynamic parameters after adjusting for significant confounding factors. RESULTS: In the multivariable analysis, after adjustment for hypertensive disorders of pregnancy and smoking, FGR was associated with a 14% increase in maternal total vascular resistance, 16% reduction in cardiac output, 13% reduction in left ventricular mass and 11% reduction in heart rate; similar results were observed for the corresponding indexed parameters. Hypertensive disorders of pregnancy in the absence of FGR were associated with a 25% increase in total vascular resistance, 16% increase in left ventricular mass and 14% reduction in diastolic function; similar results were observed for the corresponding indexed parameters. CONCLUSION: FGR is significantly and independently associated with several maternal hemodynamic parameters, even after adjustment for major confounding factors, such as hypertensive disorders of pregnancy. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Clinical and epidemiological characteristics of infectious keratitis in Paraguay.

To describe the clinical and epidemiological characteristics of patients with severe infectious keratitis in Asunción, Paraguay between April 2009 and September 2011. All patients with the clinical diagnosis of severe keratitis (ulcer ≥2 mm in size and/or central location) were included. Empiric treatment consisted of topical antibiotics and antimycotics; in cases of advanced keratitis, fortified antibiotics were used. After microbiological analysis, treatment was changed if indicated. In total 48 patients (62.5 % males, 25 % farmers) were included in the analysis. A central ulcer was found in 81.3 % (n = 39). The median delay between onset of symptoms and time of first presentation at our institution was 7 days (range 1-30 days). Fungal keratitis was diagnosed in 64.5 % (n = 31) of patients, of which Fusarium sp. (n = 17) was the most common. Twenty-one patients (43.8 %) reported previous trauma to the eye. The globe could be preserved in all cases. While topical therapy only was sufficient in most patients, a conjunctival flap was necessary in six patients suffering from fungal keratitis. The high rate of fungal keratitis in this series is remarkable, and microbiological analysis provided valuable information for the appropriate treatment. In this setting, one has to be highly suspicious of fungal causes of infectious keratitis.

Characterizing pearls structures using X-ray phase-contrast and neutron imaging: a pilot study.

Some cultured and natural pearls can be reliably distinguished by visual inspection and by the use of lens and microscope. However, assessing the origin of the pearls could be not straightforward since many different production techniques can now be found in the pearl market, for example in salt or freshwater environments, with or without a rigid nucleus. This wide range of products requires the use of new effective scientific techniques. Indeed, X-ray radiography has been used by gemologists since last century as the only safe and non-destructive way to visually inspect the interior of a pearl, and recently, also X-ray computed micro-tomography was used to better visualize the inner parts of the gems. In this study we analyzed samples of natural and cultured pearls by means of two non-destructive techniques: the X-ray Phase-Contrast Imaging (PCI) and the Neutron Imaging (NI). PCI and NI results will be combined for the first time, to better visualize the pearls internal morphology, thus giving relevant indications on the pearl formation process.

Inducible expression of calcyclin, a gene with strong homology to S-100 protein, during neuroblastoma cell differentiation and its prevalent expression in Schwann-like cell lines.

Calcyclin gene expression was evaluated in different neuroblastoma cell lines and during neuronal differentiation induced by retinoic acid. Calcyclin gene expression was more frequently detected in epithelial-type or Schwann-like cells rather than in neuroblastic cells. This result indicates an increase of G1 cell fraction, which may explain the limited growth potential usually observed for these cells. LAN-5 cell (neuronal type) differentiation experiments showed that calcyclin gene is detectable after 4 days of retinoic acid treatment, which induces G1 phase accumulation (as detected by cytofluorometric analysis), and cell growth arrest. Otherwise, neither block of cell proliferation by 0.5% fetal calf serum medium nor addition of 15% fresh fetal calf serum after cell arrest induce calcyclin expression. The increase of calcyclin mRNA levels during cell differentiation shows that calcyclin gene expression is associated with neuronal differentiation. This bivalent role of the calcyclin gene, which is normally expressed in the G1 phase of the cell cycle but also expressed during retinoic acid-induced neuroblastoma cell differentiation, suggests that (at least in neuroblastoma cells) the gene is subject to a complex transcriptional regulation.

Maternal cardiac deceleration capacity: a novel insight into maternal autonomic function in pregnancies complicated by hypertensive disorders and intrauterine growth restriction.

OBJECTIVE: To explore maternal cardiac deceleration capacity (DC), a marker of autonomic function derived from electrocardiographic (ECG) signals, in pregnancies complicated by intrauterine growth restriction (IUGR) and hypertensive disorders of pregnancy (HDP) associated to IUGR (HDP-IUGR) or to appropriate for gestational age fetal growth (HDP-AGAf). METHODS: Prospective single center case-control study conducted at Buzzi Children's Hospital, Milan. Maternal ECGs were analyzed by Phase Rectified Signal Averaging (PRSA) method to obtain cardiac DC in women with: HDP-IUGR, HDP-AGAf, severe-IUGR, mild-IUGR and uncomplicated pregnancies. IUGR was defined as abdominal circumference <5th centile; severe-IUGR was associated with umbilical artery Doppler pulsatility index >2 standard deviations. Non-parametric tests were adopted. RESULTS: 269 women were recruited. Women with HDP-IUGR (n=35) showed significantly higher cardiac DC compared both to controls (n=141) (p=0.003) and women with HDP-AGAf (n=18) (p=0.01). Women with severe-IUGR (n=14) showed significantly higher DC than controls (p=0.01). Women with mild-IUGR (n=61) as well as women with HDP-AGAf showed no differences in DC compared to controls (both p=0.3). CONCLUSIONS: Women with pregnancy complicated by severe placental failure, such as HDP-IUGR and severe IUGR, show significant autonomic alterations, as indicated by elevated cardiac DC. On the contrary, pregnancy complications such as HDP-AGAf and mild IUGR show no impact on maternal autonomic balance. We present a new approach to explore maternal autonomic cardiovascular regulation that might reflect the severity of placental vascular insufficiency.

The expression of metalloproteinase-2, -9, and -14 and of tissue inhibitors-1 and -2 is developmentally modulated during osteogenesis in vitro, the mature osteoblastic phenotype expressing metalloproteinase-14.

During osteogenesis, in vitro, of tibial-derived rat osteoblasts (ROB) and derived clones, changes occur in the interactions of mature osteoblasts with the endogenous extracellular matrix (ECM) and these culminate in the formation of tridimensional nodules, which become sites of mineral deposition. We investigated if these changes might be mediated by remodeling of ECM, and we focused our study on the neutral metalloproteinases (MMPs), known agents of matrix remodeling, and on their tissue inhibitors (TIMPs). We report that during in vitro differentiation, osteoblasts express the secreted MMP-2 and -9 and the membrane gelatinase MMP-14. These, along with the tissue inhibitors TIMP-1 and -2, are developmentally regulated according to the maturation stage of osteoblasts. Their levels change in a similar association with osteoblast phenotypic maturation in different populations of ROB, which take different times to complete osteogenesis in vitro. MMP-14 expression coincides in both cell populations with the mature osteoblastic phenotype and is localized in the cells forming nodules. MMP-2 and -9 are expressed diffusely in the osteoblast population. Developmentally associated changes in the activation of MMP-2 are detected, associated in their timing with the expression of MMP-14 in both populations of ROB, and MMP-14 activates pro-MMP-2 in vitro. Expression of messenger RNAs (mRNAs) for the three MMPs increases up to the time of nodule formation. At this stage, TIMP-1 mRNA levels are lowest. TIMP-2 mRNA decreases throughout osteogenesis. In situ hybridization in 7-day-old rat tibias shows the strongest expression of MMP-14 among osteogenic cells, in lining osteoblasts on the newly formed trabeculae under the growth plate, and on the endosteal surface of cortical bone. Our data support the concept that the developmentally regulated expression of MMP-14 triggers localized proteolysis within the osteogenic population, concomitant in vitro to nodule formation.

Expression of protein kinase C-alpha (PKC-alpha) and MYCN mRNAs in human neuroblastoma cells and modulation during morphological differentiation induced by retinoic acid.

It is known that PKC is differently expressed in brain and the peripheral nervous system and is involved in cellular differentiation. We have analyzed 9 human neuronal-derived crest-cell lines for PKC-alpha mRNA. Seven out of nine expressed 9.0 kb and 4.0 kb PKC-alpha mRNAs, but three had high level of 9.0 kb transcription. The different expression of the two messenger RNAs may result from alternative splicing and a different degree of cell maturation. The same cell lines were studied for MYCN gene expression. A possible relation between the two genes is discussed. One cell line expressing high levels of both PKC-alpha mRNA was treated with 10(-5) M retinoic acid (RA). The expression of both messenger RNAs was suppressed when the cells achieved a morphological differentiation and showed neurite-like processes. A decrease of PKC-alpha gene expression was associated to down regulation of MYCN mRNA. These preliminary results suggest that PKC suppression of PKC-alpha mRNA is associated with reversion of the malignant phenotype.

Neurite outgrowth and cell cycle kinetic changes induced by cis-diamminedichloroplatinum II and retinoic acid in a human neuroblastoma cell line.

The aim of this study was to analyze by flow cytometry the effect of cis-diamminedichloroplatinum II (CDDP) and retinoic acid (RA) on the cell cycle of a neuroblastoma cell line (SK-N-BE (2)C NB) and to correlate the kinetic data with cell morphology. CDDP at 1 microgram/ml induced a dramatic G2 + M cell cycle phases block (nearly 200% increase with respect to control) 2 days after treatment. The G2 + M block was spontaneously reversed starting from the 4th day. The cells treated with 10 microM RA were, instead, induced to irreversibly enter the G0 + G1 phase of the cell cycle (nearly 20% increase with respect to control) 48 h after treatment. Neurite-like structures were observed for both CDDP and RA treated cells. These data suggest different cell cycle dependent molecular mechanisms and different degrees of differentiation during CDDP or RA treatment of NB cells.

Expression of histone H3 cell cycle-related gene, vimentin and MYC genes in pediatric brain tumors. A preliminary analysis showing the different malignant cell growth potential.

Eleven pediatric brain tumors were studied for the histone H3, Vimentin and MYC gene expression. H3, an S phase cell cycle-related gene (ccr), was found prevalently expressed in tumors with a high mitotic index (MI). Vimentin gene, which contributes to maintaining the cell structure but is also demonstrated to be an early responder gene to growth stimulation was found variously expressed. The different expression of Vimentin gene in the examined samples suggests the active proliferation of the tumor cells. Analysis of MYC gene expression was found increased only in a mesenchymal chondrosarcoma while in other samples MYC mRNA was undetectable. Medulloblastoma, chondrosarcoma, and choroid plexus carcinoma have high S phase H3 gene expression associated with a high MI. Differently an astrocytoma shows a low MI associated with high H3 gene expression. This first preliminary report of H3, Vimentin and MYC gene expression in brain tumors demonstrates that malignant cells are characterized by a different gene expression and different growth potentials.

MYCN amplification does not affect survival of neuroblastoma patients treated with autologous bone marrow transplantation.

From an extended series neuroblastoma cases evaluated for MYCN amplification (MNA) at the "G. Gaslini" Hospital 15 (4 with and 11 without NMA) underwent myeloablative therapy and bone marrow transplantation (MAT-ABMT). Such cases ranged in age at diagnosis from 13 months to 7 years and were followed up at least 8 months after MAT-ABMT. MNA was present in 2/10 cases dead for disease, in 0/1 cases alive with disease, and in 2/4 cases presently in complete clinical remission. This preliminary evidence would discourage to consider MNA as a marker capable of predicting the final outcome of patients with metastatic Nb.

Coordinate change of c-myc, transferrin receptor and H3 gene expression precedes induction of haemoglobin-producing cells of the leukaemia K562 cell line treated with cis-diamminedichloroplatinum (II).

Erythroid cell differentiation can be achieved in vitro by means of several chemical or natural agents. Cell differentiation is accompanied by biochemical and molecular changes which show that the inducer is active at different molecular levels. Among the chemical agents that are able to induce cell differentiation, the anticancer drugs are of great interest for the emerging study of in vitro and in vivo models for differentiation treatment. Cis-diamminedichloroplatinum II (CDDP), a crosslinking DNA compound, is usually employed at a high dosage in treating solid tumors. We have demonstrated that CDDP was also able to induce K562 cells towards erythroid differentiation. In our experiment 2.5 micrograms/ml of CDDP caused expression of alpha-globin chain gene and production of haemoglobulin. Continuous presence of the drug was not necessary to induce the cell to produce haemoglobin. Five days after CDDP treatment, the expression of c-myc oncogene had risen, while H3 histone mRNA expression fell to undetectable levels within 24 hours. Transferrin (Tf) receptor mRNA was reduced but later (about 48 hours) than c-myc and H3 messenger RNAs. The inhibition of cell proliferation was correlated both with the reduced expression of Tf receptor mRNA and low expression of the protein. However, expression of the cytoskeleton vimentin gene was only slightly affected during the time-course of the experiment. Data show that at least three phases were present in the irreversible CDDP induction of haemoglobin synthesis in the K562 cell. The erythroid differentiation was first preceded by a rise of c-myc mRNA, which probably precommits the cell towards the erythroid lineage. The mRNA levels of the cell-cycle dependent genes, H3 and Tf receptor, decreased and inhibition of DNA synthesis and loss of cell proliferation were detected. Finally, the alpha-globin chain gene was actively transcribed and the cell produced haemoglobin.

Expression of multiple drug resistance gene, MDR1, and N-myc oncogene in an Italian population of human neuroblastoma patients.

Thirty-four patients of an Italian population affected by neuroblastoma (NB) were evaluated at diagnosis for multidrug resistance gene (MDR1) and N-myc oncogene amplification. No patients showed MDR1 amplification, while extra copies of the N-myc gene were found in 9 out of 34 patients (26%). N-myc amplification was correlated (p = 0.008) with a shorter progression-free survival. RNA was purified from fresh tumor biopsies and analysed in 29 NB samples. MDR1 gene expression was found to be increased in 5 out of 29 tumor samples at onset (17%) and in 1 out of 3 at relapse, but none of them expressed both MDR1 and N-myc genes simultaneously. No correlation was found between MDR1 or N-myc genes expression and tumor progression. MDR1 mRNA transcription may occur spontaneously after onset, suggesting that certain NB tumors could be resistant to antineoplastic drugs at onset. All 5 patients showing MDR1 mRNA transcription achieved complete or partial clinical remission after polychemotherapy. This was presumably due to inclusion in the therapeutic protocol of a high dose of Cisplatin, a drug not susceptible to the effects of the MDR1 gene product. Our findings show that cells which actively transcribe for the MDR1 gene are present in several untreated NB patients. No gene amplification was detected and probably the MDR1 gene expression is regulated at the transcriptional level.

Adenoviral-mediated gene transfer to the filtering bleb in rabbits.

PURPOSE: To determine if genes can be transferred to fibroblasts in the filtering bleb using adenoviral vectors. MATERIALS AND METHODS: Twelve New Zealand albino rabbits underwent bilateral full-thickness sclerostomies. On postoperative day 1 an adenoviral vector carrying a reporter gene (lacZ) was injected into the right-eye bleb and saline was injected into the left eye bleb of each rabbit. Three rabbits were euthanized on each of the after days (days 3, 7, 14, and 21). The eyes were enucleated and tissue samples from the filtering bleb were processed for beta-galactosidase activity (a marker for lacZ gene expression) and expression of vimentin (a fibroblast marker). The median number of cells per high-power field with both beta-galactosidase activity and vimentin expression on days 3, 7, 14, and 21 in the right and left eyes were counted to determine adenoviral-mediated gene expression in fibroblasts. RESULTS: In the adenoviral vector-treated eyes, the median number of cells per high-power field with both beta-galactosidase and vimentin expression on days 3, 7, 14, and 21 was 83,100, 1, and 0, respectively. No beta-galactosidase activity was noted in the saline-treated eyes. CONCLUSIONS: Adenoviral vectors can transfer genes to fibroblasts in filtering blebs after glaucoma surgery. The peak efficiency of gene transfer to fibroblasts occurred 7 days after glaucoma surgery. These studies show a potential for genetic manipulation of fibroblast activity in filtering blebs after glaucoma surgery.

A novel approach in personal identification from tissue samples undergone different processes through STR typing.

Short tandem repeats (STRs) or microsatellites have been recognized worldwide as a powerful tool for human identification. They have become widely used in human identification especially in criminal cases and mass disasters. Police departments are often interested in cases where tissues are already decomposed and only do bones remain to let them perform laboratory analyses. Bone is the most resistant tissue in animal body to time depending degradation and putrefaction, but it is often hard to extract DNA from it because of its highly mineralized structure, which makes DNA extraction and/or amplification hard to carry out. We have performed human nuclear DNA extraction and STR typing in three different cases, on bones and bone fragments from long time dead persons found buried, in the sea, almost completely burnt and whose tissues were already decomposed. We report these caseworks as we would like to show how forensic scientists are improving their skill in identifying people whose corps have undergone several kinds of processes, even independently on the time passed and the level of putrefaction of their tissues.

DNA extraction from blood determination membrane card test.

Among the several techniques applied in our laboratory, in order to establish human nature of blood specimens we perform an immunodiagnostic analysis through a specific membrane test card, which is usually utilized as a fast screening test for human haemoglobin in diagnostic checks. This test works with a double-step method based upon solubilization of the sample in a proper buffer and its migration on a membrane monoclonal and polyclonal antibodies specific against haemoglobin are linked to. The complexes haemoglobin-antibodies migrate through the membrane and are revealed by an immunoreactive line turning reddish-coloured to show that the reaction took place and the system works. As this test can recognize both the whole haemoglobin molecule and the degraded one, it is usually exploited in forensic sciences to detect kind and species of blood specimens related to forensic case works. However, as a consequence of the small amounts of blood in the samples collected from the crime scene we often cannot perform this kind of analysis in order to save the sample and be able to genotype it. Then, we investigated the possibility to purify DNA from the blood specimens already dissolved in the extraction buffer and perform STR typing on it with several methods and after different storage conditions and time.

Experimental procedures comparing the activity of different Taq polymerases.

Forensic investigations involve several scientific branches among which biological analyses are much more frequently requested as a consequence of their importance and great versatility towards most of the traces found on the crime scene. Biological analyses are lead in subsequent steps: extraction, amplification and STR typing of the specimens collected on the crime scene. All of these techniques have been modified from original protocols according to the kind of sample to process. A critical point in our analysis is trying to amplify small amounts of DNA extracted from decomposed tissues or objects, small biological traces have been left on, with high fidelity and account. That's why we have decided to settle on an experimental procedure aimed to find the best DNA polymerase according to our purposes. We have tested different Taq polymerases on the same known DNA sample at several dilutions and have compared quality and amount of amplified DNA in order to appreciate the amplifying capability of each enzyme. These data have been analyzed as a function of the technical properties of each engineered Taq polymerase and results are shown in details.

Single sperm cell isolation by laser microdissection.

In 1996, Van Oorschot and Jones firstly reported in scientific correspondence that short tandem repeat (STR) profiles could be obtained from cells left on different objects. Since then, forensic scientists have focused their efforts in isolating single cells as it can be extremely helpful in solving case works where sexual violence was concerned. Laser microdissection is a micromanipulation procedure allowing to cut off precisely the cells of interest from tissue samples or smears by a laser beam fitted with an optical microscope. We have harvested single sperm cells by laser microdissection using a Leica AS LMD (Leica Microsystems, Germany); laser setting, pulse laser intensity and laser alignment as well as recovery of the specimen have been properly fitted to the samples we were dealing with. Different tissue preservation, fixation, histological staining (Papanicolau, Nuclear Fast Red-Picroindigocarmine) methods and number of harvested cells for each sample have been evaluated as well. Finally, the genotype of sperm cells has been determined by STR typing, evaluating the sensibility of this forensic technique according to instrumental and biological above-mentioned variables.

Laser microdissection and DNA typing of cells from single hair follicles.

Isolation and identification of single cells from tissue samples or smears assume a great relevance in pathological and forensic applications; in this latter field, the possibility to identify a specific genetic profile can be obtained by short tandem repeat (STR) typing, allowing to achieve a scientific proof important in law courts. It is well known that DNA extraction may be performed from several tissue fragments, blood traces, spermatozoa as well as telogen hair. However, in the last case, few follicle cells are coupled to a great amount of keratin reducing the efficiency of DNA amplification. Recently, the introduction of laser microdissection technique has greatly improved the capability to select single cells without any cross-contamination. In the present report, we have performed a laser microdissection using a Leica AS LMD (Leica Microsystems, Germany), utilized on cutting the telogen hair in order to exclusively collect the lower part of the follicle and reduce keratin contamination. In this way we can accurately extract an adequate amount of DNA, successfully typed by STR profile.

[Fecal chymotrypsin and steatorrhea in chronic pancreatitis]. / Chimotripsina fecale e steatorrea nella pancreatite cronica.

The aim of the present study was to evaluate in 56 patients (48 M, 8 F) with chronic pancreatitis: a) the diagnostic validity of fecal chymotrypsin (FCT) assay, performed both on random samples and from previously homogenized samples collected over 3 days; b) the correlation between chymotrypsin and fecal fat excretion. CTF was measured using Kaspar's colorimetric method and fecal fats using the Van de Kamer method. Mean values of chymotrypsin measured on random samples were very similar to those measured on previously homogenized feces, 17.9 +/- 16.7 U/g vs 17.1 +/- 15.3 U/g respectively. There was a highly significant correlation between these values (r = 0.77 p less than 0.0003) and a highly significant inverse correlation between fecal fat and chymotrypsin excretion, both when the latter was measured on random and on previously homogenized samples (p less than 0.0001). FCT assay was fairly good sensitive (54%) for the whole group of patients with chronic pancreatitis, but very good (91%) for the group of patients with steatorrhea. The results show that the fecal chymotrypsin assay on random fecal samples is as valid as that carried out on homogenized feces and that there is a good correlation between fecal chymotrypsin values and steatorrhea of pancreatic origin.