Investigation of healthy and infected newborn saliva: the role of proteins and lipids as potential inflammatory diagnostic tools

We have carried out a discovery proteomics investigation aimed at identifying disease biomarkers present in saliva, and, more specifically, early biomarkers of inflammation. The proteomic characterization of saliva is possible due to the straightforward and non-invasive sample collection that allows repetitive analyses for pharmacokinetic studies. These advantages are particularly relevant in the case of newborn patients. The study was carried out with samples collected during the first 48 hours of life of the newborns according to an approved Ethic Committee procedure. In particular, the salivary samples were collected from healthy and infected (n=1) newborns. Proteins were extracted through cycles of sonication, precipitated in ice cold acetone, resuspended and resolved by 2D-electrophoresis. MALDI TOF/TOF mass spectrometry analysis was performed for each spot obtaining the proteins’ identifications. Then we compared healthy newborn salivary proteome and an infected newborn salivary proteome in order to investigate proteins differently expressed in inflammatory condition. In particular the protein alpha-1-antitrypsin (A1AT), correlated with inflammation, was detected differently expressed in the infected newborn saliva. Therefore, in the second part of the project we aimed to develop a robust LC-MS based method that identifies and quantifies this inflammatory protein within saliva that might represent the first relevant step to diagnose a condition of inflammation with a no-invasive assay. The same LC-MS method is also useful to investigate the presence of the F allelic variant of the A1AT in biological samples, which is correlated with the onset of pulmonary diseases. In the last part of the work we analysed newborn saliva samples in order to investigate how phospholipids and mediators of inflammation (eicosanoids) are subject to variations under inflammatory conditions and a trend was observed in lysophosphatidylcholines composition according to the inflammatory conditions.

Pulse oximetry screening for congenital heart defects in newborn infants:an evaluation of acceptability to mothers

Background: Introducing neonatal screening procedures may not be readily accepted by parents and may increase anxiety. The acceptability of pulse oximetry screening to parents has not been previously reported. Objective: To assess maternal acceptability of pulse oximetry screening for congenital heart defects and to identify factors predictive of participation in screening. Design and setting: A questionnaire was completed by a cross-sectional sample of mothers whose babies were recruited into the PulseOx Study which investigated the test accuracy of pulse oximetry screening. Participants: A total of 119 mothers of babies with false-positive (FP) results, 15 with true-positive and 679 with true-negative results following screening. Main outcome measures: Questionnaires included measures of satisfaction with screening, anxiety, depression and perceptions of test results. Results: Participants were predominantly satisfied with screening. The anxiety of mothers given FP results was not significantly higher than that of mothers given true-negative results (median score 32.7 vs 30.0, p=0.09). White British/Irish mothers were more likely to participate in screening, with a decline rate of 5%; other ethnic groups were more likely to decline with the largest increase in declining being for Black African mothers (21%, OR 4.6, 95% CI 3.8 to 5.5). White British mothers were also less anxious (p<0.001) and more satisfied (p<0.001) than those of other ethnicities Conclusions: Pulse oximetry screening was acceptable to mothers and FP results were not found to increase anxiety. Factors leading to differences in participation and satisfaction across ethnic groups need to be identified so that staff can support parents appropriately.

Pulse oximetry as a screening test for congenital heart defects in newborn infants: a test accuracy study with evaluation of acceptability and cost-effectiveness

Background: Screening for congenital heart defects (CHDs) relies on antenatal ultrasound and postnatal clinical examination; however, life-threatening defects often go undetected. Objective: To determine the accuracy, acceptability and cost-effectiveness of pulse oximetry as a screening test for CHDs in newborn infants. Design: A test accuracy study determined the accuracy of pulse oximetry. Acceptability of testing to parents was evaluated through a questionnaire, and to staff through focus groups. A decision-analytic model was constructed to assess cost-effectiveness. Setting: Six UK maternity units. Participants: These were 20,055 asymptomatic newborns at = 35 weeks’ gestation, their mothers and health-care staff. Interventions: Pulse oximetry was performed prior to discharge from hospital and the results of this index test were compared with a composite reference standard (echocardiography, clinical follow-up and follow-up through interrogation of clinical databases). Main outcome measures: Detection of major CHDs – defined as causing death or requiring invasive intervention up to 12 months of age (subdivided into critical CHDs causing death or intervention before 28 days, and serious CHDs causing death or intervention between 1 and 12 months of age); acceptability of testing to parents and staff; and the cost-effectiveness in terms of cost per timely diagnosis. Results: Fifty-three of the 20,055 babies screened had a major CHD (24 critical and 29 serious), a prevalence of 2.6 per 1000 live births. Pulse oximetry had a sensitivity of 75.0% [95% confidence interval (CI) 53.3% to 90.2%] for critical cases and 49.1% (95% CI 35.1% to 63.2%) for all major CHDs. When 23 cases were excluded, in which a CHD was already suspected following antenatal ultrasound, pulse oximetry had a sensitivity of 58.3% (95% CI 27.7% to 84.8%) for critical cases (12 babies) and 28.6% (95% CI 14.6% to 46.3%) for all major CHDs (35 babies). False-positive (FP) results occurred in 1 in 119 babies (0.84%) without major CHDs (specificity 99.2%, 95% CI 99.0% to 99.3%). However, of the 169 FPs, there were six cases of significant but not major CHDs and 40 cases of respiratory or infective illness requiring medical intervention. The prevalence of major CHDs in babies with normal pulse oximetry was 1.4 (95% CI 0.9 to 2.0) per 1000 live births, as 27 babies with major CHDs (6 critical and 21 serious) were missed. Parent and staff participants were predominantly satisfied with screening, perceiving it as an important test to detect ill babies. There was no evidence that mothers given FP results were more anxious after participating than those given true-negative results, although they were less satisfied with the test. White British/Irish mothers were more likely to participate in the study, and were less anxious and more satisfied than those of other ethnicities. The incremental cost-effectiveness ratio of pulse oximetry plus clinical examination compared with examination alone is approximately £24,900 per timely diagnosis in a population in which antenatal screening for CHDs already exists. Conclusions: Pulse oximetry is a simple, safe, feasible test that is acceptable to parents and staff and adds value to existing screening. It is likely to identify cases of critical CHDs that would otherwise go undetected. It is also likely to be cost-effective given current acceptable thresholds. The detection of other pathologies, such as significant CHDs and respiratory and infective illnesses, is an additional advantage. Other pulse oximetry techniques, such as perfusion index, may enhance detection of aortic obstructive lesions.

The impact of a father's presence during newborn resuscitation:a qualitative interview study with healthcare professionals

Objective: To explore healthcare professionals’ experiences around the time of newborn resuscitation in the delivery room, when the baby’s father was present. Design: A qualitative descriptive, retrospective design using the critical incident approach. Tape-recorded semistructured interviews were undertaken with healthcare professionals involved in newborn resuscitation. Participants recalled resuscitation events when the baby’s father was present. They described what happened and how those present, including the father, responded. They also reflected upon the impact of the resuscitation and the father’s presence on themselves. Participant responses were analysed using thematic analysis. Setting: A large teaching hospital in the UK. Participants: Purposive sampling was utilised. It was anticipated that 35–40 participants would be recruited. Forty-nine potential participants were invited to take part. The final sample consisted of 37 participants including midwives, obstetricians, anaesthetists, neonatal nurse practitioners, neonatal nurses and paediatricians. Results: Four themes were identified: ‘whose role?’ ‘saying and doing’ ‘teamwork’ and ‘impact on me’. While no-one was delegated to support the father during the resuscitation, midwives and anaesthetists most commonly took on this role. Participants felt the midwife was the most appropriate person to support fathers. All healthcare professional groups said they often did not know what to say to fathers during prolonged resuscitation. Teamwork was felt to be of benefit to all concerned, including the father. Some paediatricians described their discomfort when fathers came to the resuscitaire. None of the participants had received education and training specifically on supporting fathers during newborn resuscitation. Conclusions: This is the first known study to specifically explore the experiences of healthcare professionals of the father’s presence during newborn resuscitation. The findings suggest the need for more focused training about supporting fathers. There is also scope for service providers to consider ways in which fathers can be supported more readily during newborn resuscitation.

Activation of protein kinase B by adenosine A1 and A3 receptors in newborn rat cardiomyocytes

It is well established that adenosine receptors are involved in cardioprotection and that protein kinase B (PKB) is associated with cell survival. Therefore, in this study we have investigated whether adenosine receptors (A1, A2A and A3) activate PKB by Western blotting and determined the involvement of phosphatidylinositol 3-kinase (PI-3K)/PKB in adenosine-induced preconditioning in cultured newborn rat cardiomyocytes. Adenosine (non-selective agonist), CPA (A1 selective agonist) and Cl-IB-MECA (A(3) selective agonist) all increased PKB phosphorylation in a time- and concentration-dependent manner. The combined maximal response to CPA and Cl-IB-MECA was similar to the increase in PKB phosphorylation induced by adenosine alone. CGS 21680 (A2A selective agonist) did not stimulate an increase in PKB phosphorylation. Adenosine, CPA and Cl-IB-MECA-mediated PKB phosphorylation were inhibited by pertussis toxin (PTX blocks G(i)/G(o)-protein), genistein (tyrosine kinase inhibitor), PP2 (Src tyrosine kinase inhibitor) and by the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG 1478. The PI-3K inhibitors wortmannin and LY 294002 blocked A(1) and A(3) receptor-mediated PKB phosphorylation. The role of PI-3K/PKB in adenosine-induced preconditioning was assessed by monitoring Caspase 3 activity and lactate dehydrogenase (LDH) release induced by exposure of cardiomyocytes to 4 h hypoxia (0.5% O2) followed by 18 h reoxygenation (HX4/R). Pre-treatment with wortmannin had no significant effect on the ability of adenosine-induced preconditioning to reduce the release of LDH or Caspase 3 activation following HX4/R. In conclusion, we have shown for the first time that adenosine A1 and A3 receptors trigger increases in PKB phosphorylation in rat cardiomyocytes via a G1/G0-protein and tyrosine kinase-dependent pathway. However, the PI-3K/PKB pathway does not appear to be involved in adenosine-induced cardioprotection by preconditioning Adenosine A1 receptor .

Synthesis and characterization of silica-supported L-Lysine-based dendritic branches

Two series of novel modified silicas have been prepared in which individual dendritic branches have been attached to aminopropylsilica using standard peptide coupling methodology. The dendritic branches are composed of enantiomerically pure l-lysine building blocks, and hence, the modified silicas have the potential to act as chiral stationary phases in chromatography. In one series of modified silicas, the surface of the dendritic branch consists of Boc carbamate groups, whereas the other has benzoyl amide surface groups. Different coupling reagents have been investigated in order to maximize the loading onto the solid phase. The new supported dendritic materials have been fully characterized with properties of the bulk material determined by elemental analysis, 13C NMR, and IR spectroscopy, whereas XPS provides important information about the surface of the modified silica exposed to the incident X-rays, the key region in which potential chromatographic performance of these materials will take place. Although the bulk analyses indicate that loading of the dendritic branch onto silica decreases with increasing dendritic generation (and consequently steric bulk), XPS indicates that the optimum surface coverage is actually obtained at the second generation of dendritic growth.

Compartmentalization of the MAPK scaffold protein KSR1 modulates synaptic plasticity in hippocampal neurons

ERK1/2 is required for certain forms of synaptic plasticity, including the long-term potentiation of synaptic strength. However, the molecular mechanisms regulating synaptically localized ERK1/2 signaling are poorly understood. Here, we show that the MAPK scaffold protein kinase suppressor of Ras 1 (KSR1) is directly phosphorylated by the downstream kinase ERK1/2. Quantitative Western blot analysis further demonstrates that expression of mutated, feedback-deficient KSR1 promotes sustained ERK1/2 activation in HEK293 cells in response to EGF stimulation, compared to a more transient activation in control cells expressing wild-type KSR1. Immunocytochemistry and confocal imaging of primary hippocampal neurons from newborn C57BL6 mice further show that feedback phosphorylation of KSR1 significantly reduces its localization to dendritic spines. This effect can be reversed by tetrodotoxin (1 μM) or PD184352 (2 μM) treatment, further suggesting that neuronal activity and phosphorylation by ERK1/2 lead to KSR1 removal from the postsynaptic compartment. Consequently, electrophysiological recordings in hippocampal neurons expressing wild-type or feedback-deficient KSR1 demonstrate that KSR1 feedback phosphorylation restricts the potentiation of excitatory postsynaptic currents. Our findings, therefore, suggest that feedback phosphorylation of the scaffold protein KSR1 prevents excessive ERK1/2 signaling in the postsynaptic compartment and thus contributes to maintaining physiological levels of synaptic excitability. © FASEB.

Human Synaptic Plasticity Gene Expression Profile and Dendritic Spine Density Changes in HIV-Infected Human CNS Cells: Role in HIV-Associated Neurocognitive Disorders (HAND)

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT2 Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.

The Immunomodulatory Role of Alcohol on HIV-Infected Monocyte-Derived Dendritic Cells

Alcohol is known to induce inflammation in the presence of the human immunodeficiency virus (HIV). In our previous studies, we revealed that alcohol induces cannabinoid receptors which play a role in the regulation of inflammatory cytokine production in monocyte-derived dendritic cells (MDDC). However, the ability of alcohol to alter MDDC function during HIV infection has not been clearly elucidated yet. To study the potential impact of alcohol on HIV-infected MDDC (confirmed by p24 ELISA), monocytes were isolated from commercially available buffy coats and cultured for 7 days with GM-CSF and IL-4. MDDC were infected with HIV- 1Ba-L and treated with different concentrations of alcohol (0.1% band 0.2%) for 4-7 days. MDDC phenotype, endocytosis, cytokine production, and ability to transmit HIV to T cells were analyzed. Uninfected CD4+ T cells were co-cultured for 7 days with either infected/treated MDDC or the supernatants from infected/treated MDDC. Inflammatory cytokine arrays were performed using supernatants from HIV-infected MDDC treated with alcohol. Results showed that HIV positive MDDC treated with alcohol had higher levels of infection compared to untreated HIV positive controls. CD4+ T cells exposed to HIV-infected MDDC acquired 100-fold higher levels of p24 compared to CD4+ T cells exposed to only supernatants. CD4+ T cells exposed to HIV-infected and alcohol-treated MDDC had higher levels of infection compared to controls. Cytokine array data show dysregulation of cytokine production by alcohol. In addition, MDDC phenotype and endocytic capacity were altered in the alcohol treated MDDC. Our results indicate a crucial role of MDDC in HIV transmission to T cells and provide insights into the inflammatory role alcohol exerts on dendritic cell function in the context of HIV infection. Supported by the National Institute on Alcohol Abuse and Alcoholism award R00AA021264, the National Institute on Drug Abuse award R01DA034547, and the Institute on NeuroImmune Pharmacology at FIU.