Prediction of cardiovascular risk in rheumatoid arthritis: performance of original and adapted SCORE algorithms.

OBJECTIVES: Predictive performance of cardiovascular disease (CVD) risk calculators appears suboptimal in rheumatoid arthritis (RA). A disease-specific CVD risk algorithm may improve CVD risk prediction in RA. The objectives of this study are to adapt the Systematic COronary Risk Evaluation (SCORE) algorithm with determinants of CVD risk in RA and to assess the accuracy of CVD risk prediction calculated with the adapted SCORE algorithm. METHODS: Data from the Nijmegen early RA inception cohort were used. The primary outcome was first CVD events. The SCORE algorithm was recalibrated by reweighing included traditional CVD risk factors and adapted by adding other potential predictors of CVD. Predictive performance of the recalibrated and adapted SCORE algorithms was assessed and the adapted SCORE was externally validated. RESULTS: Of the 1016 included patients with RA, 103 patients experienced a CVD event. Discriminatory ability was comparable across the original, recalibrated and adapted SCORE algorithms. The Hosmer-Lemeshow test results indicated that all three algorithms provided poor model fit (p<0.05) for the Nijmegen and external validation cohort. The adapted SCORE algorithm mainly improves CVD risk estimation in non-event cases and does not show a clear advantage in reclassifying patients with RA who develop CVD (event cases) into more appropriate risk groups. CONCLUSIONS: This study demonstrates for the first time that adaptations of the SCORE algorithm do not provide sufficient improvement in risk prediction of future CVD in RA to serve as an appropriate alternative to the original SCORE. Risk assessment using the original SCORE algorithm may underestimate CVD risk in patients with RA.

Duhamel versus transanal endorectal pull through (TERPT) for the surgical treatment of Hirschsprung's disease.

For the surgical treatment of Hirschsprung's disease, several surgical techniques are used to resect the distal aganglionic colon. Two frequently used techniques are the Duhamel procedure and the transanal endorectal pull-through procedure. During the '8th Pediatric Colorectal Course' in Nijmegen, November 2015, a workshop was organized to share experiences of both techniques by several experts in the field and to discuss (long term) outcomes. Specifically, the objective of the meeting was to discuss the main controversies in relation to the technical execution of both procedures in order to make an initial assessment of the limitations of available evidence for clinical decision-making and to formulate a set of preliminary recommendations for current clinical care and future research.

Performance of four current risk algorithms in predicting cardiovascular events in patients with early rheumatoid arthritis.

OBJECTIVE: This study was undertaken to assess the predictive ability of 4 established cardiovascular (CV) risk models for the 10-year risk of fatal and non-fatal CV diseases in European patients with rheumatoid arthritis. METHODS: Prospectively collected data from the Nijmegen early rheumatoid arthritis (RA) inception cohort was used. Discriminatory ability for CV risk prediction was estimated by the area under the receiver operating characteristic curve. Calibration was assessed by comparing the observed versus expected number of events using Hosmer-Lemeshov tests and calibration plots. Sensitivity and specificity were calculated for the cut-off values of 10% and 20% predicted risk. RESULTS: Areas under the receiver operating characteristic curve were 0.78-0.80, indicating moderate to good discrimination between patients with and without a CV event. The CV risk models Systematic Coronary Risk Evaluation (SCORE), Framingham risk score (FRS) and Reynolds risk score (RRS) primarily underestimated CV risk at low and middle observed risk levels, and mostly overestimated CV risk at higher observed risk levels. The QRisk II primarily overestimated observed CV risk. For the 10% and 20% cut-off values used as indicators for CV preventive treatment, sensitivity ranged from 68-87% and 40-65%, respectively and specificity ranged from 55-76% and 77-88%, respectively. Depending on the model, up to 32% of observed CV events occurred in patients with RA who were classified as low risk (<10%) for CV disease. CONCLUSIONS: Established risk models generally underestimate (Systematic Coronary Risk Evaluation score, Framingham Risk Score, Reynolds risk score) or overestimate (QRisk II) CV risk in patients with RA.

Cumulative pregnancy rates after sequential treatment with modified natural cycle IVF followed by IVF with controlled ovarian stimulation.

BACKGROUND: In modified natural cycle IVF (MNC-IVF), treatment is aimed at using the one follicle that spontaneously develops to dominance, using a GnRH-antagonist together with gonadotrophins in the late follicular phase only. The MNC-IVF is of interest because of its low-risk and patient-friendly profile. The effect of application of MNC-IVF preceding standard IVF with ovarian stimulation on overall results is unknown. METHODS: This single-center cohort study provides follow-up of an earlier study in which nine cycles of MNC-IVF were offered to 268 patients. Ongoing pregnancy rates and live birth rates, as well as time-to-pregnancy after controlled ovarian stimulation-IVF (COS-IVF) following MNC-IVF, were evaluated. RESULTS: Actual observed cumulative ongoing pregnancy rates and live birth rates after sequential treatment with MNC-IVF followed by COS-IVF were 51.5 (95% CI: 45.4-57.6) and 50.0% (95% CI: 43.9-56.1) per patient, of which 8.0 and 6.7% were twins. Median time to ongoing pregnancy was 28.8 weeks. Including treatment-independent pregnancies, cumulative ongoing pregnancy rate was 56.7% (95% CI: 50.7-62.8). CONCLUSIONS: Sequential treatment with MNC-IVF followed by COS-IVF does not appear to compromise overall success rates, while twin pregnancy rate is low. Because of its patient-friendly and low-risk profile, it seems appropriate to perform MNC-IVF preceding COS-IVF.

Virus fitness: concept, quantification, and application to HIV population dynamics.

Viral fitness has been broadly studied during the past three decades, mainly to test evolutionary models and population theories difficult to analyze and interpret with more complex organisms. More recent studies, however, are focused in the role of fitness on viral transmission, pathogenesis, and drug resistance. Here, we used human immunodeficiency virus (HIV) as one of the most relevant models to evaluate the importance of viral quasispecies and fitness in HIV evolution, population dynamics, disease progression, and potential clinical implications.

Postnatal Identification of Zika Virus Peptides from Saliva.

We explored the potential to diagnose Zika virus (ZIKV) infection by analyzing peptides in saliva during a convalescent phase of infection, long after resolution of acute disease. A 25-y-old woman clinically diagnosed with Zika fever in the first trimester was enrolled with her dizygotic twins for a 3-mo postnatal sample of saliva (9-mo after maternal infection). The female baby (A) had microcephaly while the male baby (B) was born healthy. Peptidomic analysis was completed by mass spectrometry (MS/MS), and ZIKV peptides were identified using the National Institutes of Health Zika Virus Resource database, then aligned and mapped to the ZIKV polyprotein to determine proteome coverage and phylogenetic studies. A total of 423 (mother), 607 (baby A), and 183 (baby B) unique ZIKV peptides were identified in saliva by MS/MS, providing a coverage of 67%, 84%, and 45%, respectively, of the entire ZIKV polyprotein (>3,400 amino acids). All peptides were aligned to other flaviviruses that are circulating in Brazil (dengue and yellow fever) to discard false-positive matches. Nine peptides identified were highly conserved to dengue virus. Alignment of a contiguous peptide sequence for mother/babies with the 74 ZIKV sequences suggested that the virus may have entered the oral cavity through the salivary glands, leading to an infection that persists into the postnatal period (vertical transmission). Furthermore, we identified 9 sequence variations that were unique to the baby with microcephaly (not found in the mother or the twin). This sequence information could provide a template for future neuropathogenic studies. A much larger sample size is required to determine whether sequence variation in the envelope protein significantly associates with microcephaly. Finally, from a public health perspective, it will be important to determine whether viral replication is still taking place after birth and whether the virus can be transmitted through salivary contact.

Interaction of retroviral reverse transcriptase with template-primer duplexes during replication.

Conversion of the single-stranded RNA of an invading retrovirus into double-stranded proviral DNA is catalyzed in a multi-step process by a single virus-coded enzyme, reverse transcriptase (RT). Achieving this requires a combination of DNA polymerase abd ribonuclease H (RNase H) activities, which are located at the amino and carboxy terminus of the enzyme, respectively. Moreover, proviral DNA synthesis requires that three structurally-distinct nucleic acid duplexes are accommodated by this enzyme, namely (a) A-form RNA (initiation of minus strand synthesis), non-A, non-B RNA/DNA hybrid (minus strand synthesis and initiation of plus strand synthesis) and B-form duplex DNA (plus strand synthesis). This review summarizes our current understanding of the manner in which retroviral RT interacts with this diverse array of nucleic acid duplexes, exploiting in many cases mutants unable to catalyze a specific event. These studies illustrate that seemingly 'simple' events such as tRNA-primed initiation of minus strand synthesis are considerably more complex, involving intermolecular tRNA-viral RNA interactions outside the primer binding site. Moreover, RNase H activity, generally thought to catalyze non-specific degradation of the RNA-DNA replicative intermediate, is required for highly specialized events including DNA strand transfer and polypurine selection. Finally, a unique structure near the center of HIV proviral DNA, the central termination sequence, serves to halt the replication machinery in a manner analogous to termination of transcription. As these highly specialized events are better understood at the molecular level, they may open new avenues of therapeutic intervention in the continuing effort to stem the progression of HIV infection and AIDS.

Involvement of C-terminal structural elements of equine infectious anemia virus reverse transcriptase in DNA polymerase and ribonuclease H activities.

In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcriptase (EIAV RT), recombinant p66 polypeptides containing a modified ribonuclease H (RNase H) domain were purified and evaluated. Defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of DNA polymerase and RNase H activities, while DNase I footprinting revealed features of replication complexes containing the truncated enzymes. Removal of alpha-helix E' and the conserved beta 5'-alphaE' "His-loop" in p66delta20 RT uncouples the RNase H activities, alters affinity for template-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, DNase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-terminal residues, which includes alpha-helix D', beta-strand 5' and alpha-Helix E', yields an enzyme with distributive DNA polymerase properties closely resembling the purified p51 subunit.

Mature reverse transcriptase (p66/p51) is responsible for low levels of viral DNA found in human immunodeficiency virus type 1 (HIV-1).

Reverse transcription of the HIV RNA genome is thought to occur in the host cell cytoplasm after viral adsorption. However, viral DNA has been isolated in cell-free virus particles. We have quantitated by polymerase chain reaction (PCR) amplification the amount of viral DNA in virions as compared to RNA. Virus produced by proviral DNA transfections of cos-7 cells or by chronically-infected H9 cells; neither of which express the cell surface CD4 receptor, contained at least 1000 times more viral RNA than DNA. In contrast, only 60 times more RNA than DNA was present in virus particles produced by transfection of Jurkat cells, which were CD4-positive and thus potentially susceptible to superinfection. Protease-defective virus, carrying only the precursor of reverse transcriptase (RT) p160gag-pol, contained virtually no detectable DNA. These results indicate that only mature RT (p66/p51) and not its precursor (p160gag-pol) is responsible for the presence of viral DNA in HIV.

Clinical correlates and molecular basis of HIV drug resistance.

It has been widely reported that zidovudine (ZDV)-resistant variants of human immunodeficiency virus type 1 (HIV-1) can be isolated from patients undergoing prolonged therapy with this drug. At the same time, treatment of HIV-infected individuals with ZDV and other forms of nucleotide therapy, including didanosine (ddI), have enabled patients to live longer than would otherwise be the case and to enjoy improved quality of life. HIV resistance to ZDV, ddI, and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising as the virus is known to replicate at high rates in infected individuals; moreover the RT that mediates transcription of proviral DNA from viral genomic RNA is known to be highly error prone. Thus, mutants of HIV-1, which possess a drug-resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term antiviral chemotherapy. This article describes a novel mutation at site 184 within the pol gene that accounts for resistance against both ddI and zalcitibine (ddC). HIV drug resistance occurs most commonly in individuals with low CD4 cell counts who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pretreatment isolates, than do viruses from patients with more-limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause-and-effect relationship exists. Because of the error-prone nature of viral RT and the fact that the HIV-1 genome can mutate efficiently, it can be anticipated that viral drug resistance may emerge for all forms of nucleotide therapy to be offered in the future. In addition, resistance may also become apparent with regard to drugs that block HIV replication by acting at sites within the viral replication cycle other than RT.

Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription.

Reverse transcription by human immunodeficiency virus (HIV) reverse transcriptase (RT) entails several distinct, early steps in the synthesis of double-stranded DNA from viral RNA templates. These steps include tRNA(lys3) priming of RNA-dependent DNA polymerization and template switching for synthesis of near full-length (-) DNA. However, HIV RT lacks the fluent processivity of other viral reverse transcriptases or cellular polymerases. Previous studies in our laboratory showed that RT reactions primed with tRNA(lys3) had higher efficiencies of template switching than those primed by synthetic oligonucleotides. To further study primer extension, pausing, and template switching, we utilized an in vitro reconstituted reverse transcription/template switching reaction consisting of HIV RNA templates, recombinant HIV RT and primer tRNA(lys3). We observed initial pause sites within the first five nucleotides of the primer terminus; these were extended efficiently to full-length (-) strong-stop DNA with prolonged incubation. Other pause sites occurred in the R/U5/PBS template adjacent to homopolymeric sequences and regions thought to be involved in secondary structure. This assay provides a sensitive means of assessing template switching at times between 10 s and 3 h. Copyright 1995 S. Karger AG, Basel

A new method to detect acrosome-reacted spermatozoa using biotinylated soybean trypsin inhibitor.

OBJECTIVE: To develop a method to detect acrosome-reacted spermatozoa on human zonae pellucidae using only commercially available reagents and without need for sperm fixation. DESIGN: Sperm head labeling with biotinylated soybean trypsin inhibitor (SBTI-biotin) was compared with results of a known method using fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin. The SBTI-biotin method was applied to sperm bound to human zonae pellucidae. SETTING AND SUBJECTS: Healthy sperm donors with normal semen characteristics were recruited by the Laboratory for Reproductive Medicine in a university medical center. MAIN OUTCOME MEASURES: Soybean trypsin inhibitor-biotin binding patterns on nonfixed spermatozoa were visualized with avidin-Texas Red. The development in time of various patterns upon induction of acrosome reaction (AR) in suspension with Ca(2+)-ionophore A23187 was noted and compared with P. sativum agglutinin FITC labeling patterns. Soybean trypsin inhibitor-biotin labeling patterns of spermatozoa bound to human zonae pellucidae were determined. RESULTS: Soybean trypsin inhibitor-biotin bound specifically, via the SBTI-moiety, to an acrosomal factor as soon as AR started. Sperm-head labeling patterns could be assigned to defined stages of the AR process. The results were highly correlated to those obtained with P. sativum agglutinin FITC. The end point of the AR in suspension and on zonae pellucidae was SBTI-biotin binding confined to the equatorial segment. CONCLUSION: The SBTI-biotin method can be used to detect nonfixed acrosome-reacted spermatozoa both in suspension and on zonae pellucidae.

Variation in semen parameters derived from computer-aided semen analysis, within donors and between donors.

The development of computer-aided semen analysis (CASA) has made it possible to study sperm motility characteristics objectively and longitudinally. In this 2-year study of 8 sperm donors, we used CASA to measure 7 semen parameters (concentration, percentage of motile spermatozoa, curvilinear velocity, average path velocity, straight-line velocity, amplitude of lateral head displacement, and beat/cross frequency). The frequency distributions of the 7 parameters in the semen samples of each donor were investigated. All parameters but one were normally distributed; concentration was distributed log-normally. Variation within individual donors and between donors was studied. Analysis of variance demonstrated that variation between donors was not explained by the longitudinal variation within individual donors. Variations in motility characteristics between donors were substantial, which may make motility characteristics of limited value as a tool for establishing fertility. Strong correlations were found between the 7 parameters, partly because by definition, motility characteristics are interdependent. Fisher's discriminant analysis demonstrated that each donor appeared to have his own set of semen characteristics and, more specifically, his own motility signature. From this data set it can be predicted that in order to find population means among sperm, it may be more efficient to measure more subjects than to increase the number of samples per subject.

Domperidone in the treatment of dyspepsia: a double-blind placebo-controlled study.

Domperidone, at a dosage of 20 mg t.d.s before meals, in a double-blind, crossover, placebo-controlled trial reduced the level of the symptoms of dyspepsia by 76% compared to a 16% reduction with placebo. This difference was statistically significant (p less than 0.001). Thirteen of the fourteen patients in the study preferred domperidone to placebo. Four patients in the active treatment period and one in the placebo complained of mild side-effects.

Engineered micro-objects as scaffolding elements in cellular building blocks for bottom-up tissue engineering approaches.

A material-based bottom-up approach is proposed towards an assembly of cells and engineered micro-objects at the macroscale. We show how shape, size and wettability of engineered micro-objects play an important role in the behavior of cells on these objects. This approach can, among other applications, be used as a tool to engineer complex 3D tissues of clinically relevant size.

Viral fitness implications of variation within an immunodominant CD8+ T-cell epitope of HIV-1.

Cytotoxic T-lymphocyte (CTL) epitopes within the HIV genome are subject to negative and positive selective pressures, the balance of which influences CTL escape at a given epitope. We investigated whether viral fitness requirements dictate conservation of the HLA-A2 restricted immunodominant epitope SLYNTVATL (SL9). Viral clones incorporating changes throughout the SL9 epitope region were compared to consensus SL9 virus in terms of replication kinetics and relative viral fitness. Constructs recapitulating in vivo SL9-CTL escape variants showed markedly little effect on replication and fitness, as did non-natural conservative mutations targeting immunologically relevant positions of the epitope. Although certain residues of the epitope were constrained by viral requirements, our research reveals that there are multiple SL9 variants that are well tolerated virologically but fail to arise in vivo. In light of this data, assumptions regarding the balance of immune and viral selective pressures on this immunodominant epitope sequence need to be reassessed.