Aberrant Expression of Light and its Associated Receptors in Systemic Lupus Erythematosus and Coeliac Disease

Members of the TNF superfamily play important roles in the development and maintenance of an effective immune response. One such member of this superfamily, LIGHT, can act as a ligand for three receptors, HVEM, LT-R and DcR3. The engagement of LIGHT and HVEM, an important secondary signal for the full activation of T cells, results in a strong Th1 type response with increased production of cytokines such as INF--. The LIGHT-LT-R pathway plays a role in the recruitment of immune cells to sites of inflammation and can induce apoptosis in certain cells. DcR3, a soluble receptor, is speculated to function in modulating LIGHT’s activity by preventing it from binding with HVEM and LT-R. Two studies published in 2001 highlighted the in vivo effects of constitutive expression of LIGHT on T cells in transgenic mice. The phenotype observed in these mice indicated that there was a loss of self-tolerance leading to systemic autoimmunity as characterised by the presence of multiple autoantibodies and inflammation of numerous organs. The aim of this thesis is to investigate the expression of LIGHT and associated receptors in systemic lupus erythematosus (SLE) and coeliac disease (CD). Based on the disease manifestations seen in LIGHT transgenic mice we speculated that patients with these conditions would have altered mRNA expression patterns of LIGHT or possibly its receptors. To achieve our aim, quantitative Real-time PCR was to be employed to perform a gene expression study using a control group of healthy volunteers as well as cohorts of CD and SLE patients. A cohort of Wegener’s granulomatosis (WG) patients was also included in this study. Real-time PCR has emerged as a powerful technique for quantifying the expression of genes. However, before embarking on a logistically complex and expensive gene expression study it was felt that gaining hands-on experience in developing Real-time PCR assays for simpler studies, where the end results could be predetermined, would yield benefits in the long-term. To this end, we chose two models to study various aspects of Real-time PCR assay design. II Cystic fibrosis is the most common lethal recessive genetic disease in Caucasian populations and thus serves as a good model for developing a Real-time PCR assay for mutation detection. After identifying insufficiencies with how mutation detection is traditionally performed, we developed a novel approach on the LightCycler instrument by combining ARMS PCR with melting curve analysis. This approach allowed better design of hybridisation probes and facilitated more than one mutation to be detected per fluorescent colour channel on the LightCycler. Our optimised assays allow the detection of the five most common mutations, accounting for 90% of mutant alleles, in approximately 35 mins, which is significantly quicker than other traditional techniques. As a model for examining the capabilities of quantitative Real-time PCR, we examined the gene dosage of peripheral myelin protein 22 (PMP22). Alterations in gene copy number of PMP22 can result in two distinct neurological diseases Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), respectively caused by a gain or loss of a copy of the PMP22 gene. In this study, we successfully developed a quantitative multiplex Real-time PCR that can diagnose CMT1A and HNPP patients with altered PMP22 gene copy numbers. Our assay provides a rapid alternative to traditional techniques used for gene dosage quantification and could be adapted for use in the diagnosis of many genetic diseases. Having gained knowledge in the use of Real-time PCR we proceeded to performing the gene expression study. During this study we have provided strong evidence that LIGHT and its associated receptors may play an important role in the pathogenesis of both SLE and CD and less so in WG. Our results demonstrated that LIGHT is elevated in both peripheral blood and the small intestine of patients with active CD. In SLE, there is a more profound dysregulation of LIGHT and associated receptors. Elevated levels of LIGHT and its three receptors, HVEM, LT-R and DcR3, were identified in the peripheral blood of our SLE patients. The WG and control cohorts showed similar levels of expression for LIGHT and its receptor indicating that these signalling pathways are not involved in its pathogenesis. III Studies we performed using P/I activated Jurkat cells demonstrated that there is good correlation between LIGHT mRNA upregulation and the appearance of soluble LIGHT protein in tissue culture supernatants. The analysis of soluble LIGHT levels using ELISA demonstrated that there was a significant elevation of soluble LIGHT in the SLE cohort also a large percentage of the CD patients were strongly positive. The WG patients showed a significant reduction in soluble LIGHT compared to the controls, which provides further proof that it is not involved in its pathogenesis. Overall, our research shows that LIGHT is upregulated in both CD and SLE and given its function in promoting a strong Th1 response it is likely to contribute in the pathogenesis of both diseases. We speculate that increased LIGHT activity in SLE may promote the development of glomerulonephritis one of the more serious clinical manifestations of the disease, which can ultimately lead to renal failure and death. CD patients have a high incidence of developing secondary autoimmune disease. We hypothesize that in addition to its immediate effects in CD pathogenesis, sustained levels of soluble LIGHT may also contribute to a breakdown of selftolerance and lead to autoimmune disease development. Further research into the expression and function of LIGHT will lead to a better understanding of the mechanisms causing these diseases. In the future, this should lead to the development of new therapies for these and related diseases

The use of cardiotonic drugs in neonates

There is a distinct lack of age-appropriate cardiotonic drugs, and adult derived formulations continue to be administered, without evidence-based knowledge on their dosing, safety, efficacy, and long-term effects. Dopamine remains the most commonly studied and prescribed cardiotonic drug in the neonatal intensive care unit (NICU), but evidence of its effect on endorgan perfusion still remains. Unlike adult and pediatric critical care, there are significant gaps in our knowledge on the use of various cardiotonic drugs in various forms of circulatory failure in the NICU

Lost in transition: a systematic review of neonatal electroencephalography in the delivery room - Are we forgetting an important biomarker for newborn brain health?

Background: Electroencephalography (EEG) monitoring is routine in neonatal intensive care units (NICUs) for detection of seizures, neurological monitoring of infants following perinatal asphyxia, and increasingly, following preterm delivery. EEG monitoring is not routinely commenced in the delivery room (DR). Objectives: To determine the feasibility of recording neonatal EEG in the DR, and to assess its usefulness as a marker of neurological well-being during immediate newborn transition. Methods: We performed a systematic stepwise search of PubMed using the following terms: infant, newborns, neonate, DR, afterbirth, transition, and EEG. Only human studies describing EEG monitoring in the first 15 min following delivery were included. Infants of all gestational ages were included. Results: Two original studies were identified that described EEG monitoring of newborn infants within the DR. Both prospective observational studies used amplitude-integrated EEG (aEEG) monitoring and found it feasible in infants >34 weeks' gestation; however, technical challenges made it difficult to obtain continuous reliable data. Different EEG patterns were identified in uncompromised newborns and those requiring resuscitation. Conclusion: EEG monitoring is possible in the DR and may provide an objective baseline measure of neurological function. Further feasibility studies are required to overcome technical challenges in the DR, but these challenges are not insurmountable with modern technology

The influence of correlation on the extreme traffic loading of bridges

Accurate traffic loading models based on measured data are essential for the accurate assessment of existing bridges. There are well-established methods for the Monte Carlo simulation of single lanes of traffic, and this can easily be extended to model the loading on bridges with two independent streams of traffic in opposing directions. However, a typical highway bridge will have multiple lanes in the same direction, and various types of correlation are evident in measured traffic. This paper analyses traffic patterns using multi-lane WIM data collected at two European sites. It describes an approach to the Monte Carlo simulation of this traffic which applies variable bandwidth kernel density estimators to empirical traffic patterns of vehicle weights, gaps and speeds. This method provides a good match with measured data for multi-truck bridge loading events, and it is shown that correlation has a small but significant effect on lifetime maximum load effects

Effects of gestational and postnatal exposure to chronic intermittent hypoxia on diaphragm muscle contractile function in the rat

Alterations to the supply of oxygen during early life presents a profound stressor to physiological systems with aberrant remodeling that is often long-lasting. Chronic intermittent hypoxia (CIH) is a feature of apnea of prematurity, chronic lung disease, and sleep apnea. CIH affects respiratory control but there is a dearth of information concerning the effects of CIH on respiratory muscles, including the diaphragm—the major pump muscle of breathing. We investigated the effects of exposure to gestational CIH (gCIH) and postnatal CIH (pCIH) on diaphragm muscle function in male and female rats. CIH consisted of exposure in environmental chambers to 90 s of hypoxia reaching 5% O2 at nadir, once every 5 min, 8 h a day. Exposure to gCIH started within 24 h of identification of a copulation plug and continued until day 20 of gestation; animals were studied on postnatal day 22 or 42. For pCIH, pups were born in normoxia and within 24 h of delivery were exposed with dams to CIH for 3 weeks; animals were studied on postnatal day 22 or 42. Sham groups were exposed to normoxia in parallel. Following gas exposures, diaphragm muscle contractile, and endurance properties were examined ex vivo. Neither gCIH nor pCIH exposure had effects on diaphragm muscle force-generating capacity or endurance in either sex. Similarly, early life exposure to CIH did not affect muscle tolerance of severe hypoxic stress determined ex vivo. The findings contrast with our recent observation of upper airway dilator muscle weakness following exposure to pCIH. Thus, the present study suggests a relative resilience to hypoxic stress in diaphragm muscle. Co-ordinated activity of thoracic pump and upper airway dilator muscles is required for optimal control of upper airway caliber. A mismatch in the force-generating capacity of the complementary muscle groups could have adverse consequences for the control of airway patency and respiratory homeostasis

Early Life Exposure to Chronic Intermittent Hypoxia Primes Increased Susceptibility to Hypoxia-Induced Weakness in Rat Sternohyoid Muscle during Adulthood

Intermittent hypoxia is a feature of apnea of prematurity (AOP), chronic lung disease, and sleep apnea. Despite the clinical relevance, the long-term effects of hypoxic exposure in early life on respiratory control are not well defined. We recently reported that exposure to chronic intermittent hypoxia (CIH) during postnatal development (pCIH) causes upper airway muscle weakness in both sexes, which persists for several weeks. We sought to examine if there are persistent sex-dependent effects of pCIH on respiratory muscle function into adulthood and/or increased susceptibility to re-exposure to CIH in adulthood in animals previously exposed to CIH during postnatal development. We hypothesized that pCIH would cause long-lasting muscle impairment and increased susceptibility to subsequent hypoxia. Within 24 h of delivery, pups and their respective dams were exposed to CIH: 90 s of hypoxia reaching 5% O2 at nadir; once every 5 min, 8 h per day for 3 weeks. Sham groups were exposed to normoxia in parallel. Three groups were studied: sham; pCIH; and pCIH combined with adult CIH (p+aCIH), where a subset of the pCIH-exposed pups were re-exposed to the same CIH paradigm beginning at 13 weeks. Following gas exposures, sternohyoid and diaphragm muscle isometric contractile and endurance properties were examined ex vivo. There was no apparent lasting effect of pCIH on respiratory muscle function in adults. However, in both males and females, re-exposure to CIH in adulthood in pCIH-exposed animals caused sternohyoid (but not diaphragm) weakness. Exposure to this paradigm of CIH in adulthood alone had no effect on muscle function. Persistent susceptibility in pCIH-exposed airway dilator muscle to subsequent hypoxic insult may have implications for the control of airway patency in adult humans exposed to intermittent hypoxic stress during early life

The Gut Microbiota Composition in Dichorionic Triplet Sets Suggests a Role for Host Genetic Factors

peer-reviewedMonozygotic and dizygotic twin studies investigating the relative roles of host genetics and environmental factors in shaping gut microbiota composition have produced conflicting results. In this study, we investigated the gut microbiota composition of a healthy dichorionic triplet set. The dichorionic triplet set contained a pair of monozygotic twins and a fraternal sibling, with similar pre- and post-natal environmental conditions including feeding regime. V4 16S rRNA and rpoB amplicon pyrosequencing was employed to investigate microbiota composition, and the species and strain diversity of the culturable bifidobacterial population was also examined. At month 1, the monozygotic pair shared a similar microbiota distinct to the fraternal sibling. By month 12 however, the profile was more uniform between the three infants. Principal coordinate analysis (PCoA) of the microbiota composition revealed strong clustering of the monozygotic pair at month 1 and a separation of the fraternal infant. At months 2 and 3 the phylogenetic distance between the monozygotic pair and the fraternal sibling has greatly reduced and by month 12 the monozygotic pair no longer clustered separately from the fraternal infant. Pulse field gel electrophoresis (PFGE) analysis of the bifidobacterial population revealed a lack of strain diversity, with identical strains identified in all three infants at month 1 and 12. The microbiota of two antibiotic-treated dichorionic triplet sets was also investigated. Not surprisingly, in both triplet sets early life antibiotic administration appeared to be a major determinant of microbiota composition at month 1, irrespective of zygosity. By month 12, early antibiotic administration appeared to no longer exert such a strong influence on gut microbiota composition. We hypothesize that initially host genetics play a significant role in the composition of an individual’s gut microbiota, unless an antibiotic intervention is given, but by month 12 environmental factors are the major determinant.This study was performed as part of the INFANTMET project (10/RD/Infantmet/MFRC/705) and was funded by the Government of Ireland's Department of Agriculture Fisheries and in part by Alimentary Pharmabiotic Centre. KM is a Teagasc Walsh Fellow. CS, RPR and PWOT are members of The Alimentary Pharmabiotic Centre, which is a Centre for Science and Technology (CSET) funded by the Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant no. 02/CE/B124 and 07/CE/B1368)

Enhanced monitoring of the preterm infant during stabilization in the delivery room

Monitoring of preterm infants in the delivery room (DR) remains limited. Current guidelines suggest that pulse oximetry should be available for all preterm infant deliveries, and that if intubated a colorimetric carbon dioxide detector should provide verification of correct endotracheal tube placement. These two methods of assessment represent the extent of objective monitoring of the newborn commonly performed in the DR. Monitoring non-invasive ventilation effectiveness (either by capnography or respiratory function monitoring) and cerebral oxygenation (near-infrared spectroscopy) is becoming more common within research settings. In this article, we will review the different modalities available for cardiorespiratory and neuromonitoring in the DR and assess the current evidence base on their feasibility, strengths, and limitations during preterm stabilization

Response: Commentary: Enhanced monitoring of the preterm infant during stabilization in the delivery room

A commentary on: Enhanced Monitoring of the Preterm Infant during Stabilization in the Delivery Room by Hutchon DJ. Front Pediatr (2016) 4:64. doi: 10.3389/fped.2016.0006