Ms Sharron Hall

Midkine (MK) is a heparin-binding growth factor, whose role as a biomarker of coronary artery disease, myocardial ischaemia and necrosis has not been well measured. This study quantified serial MK levels in patients undergoing coronary angiography (CA) and identified factors associated with MK. In this single-centre, parallel cohort study, forty patients undergoing CA had arterial samples collected prior, 10 and 20¿min after heparin administration. Four groups were examined: 1¿stable coronary artery disease (CAD) without percutaneous coronary intervention (PCI); 2¿stable CAD for elective PCI; 3¿non-ST elevation myocardial infarction (NSTEMI) with or without PCI; 4¿ST elevation myocardial infarction (STEMI) with primary PCI. Groups 1, 2 and 4 were heparin naïve, allowing assessment of the effects of myocardial necrosis between baseline levels; group 3 had received low-molecular-weight heparin. MK levels were analysed by ELISA. Median MK at baseline did not differ between groups, demonstrating that myocardial ischaemia or necrosis does not affect MK levels. Heparin administration had an immediate effect on median MK at 10¿min, showing an average 500-fold increase that is dose-dependent (R2 = 0.35, p = 0.001). Median MK levels remained elevated at 20¿min following heparin administration. Multivariate analysis showed that the estimated glomerular filtration rate (eGFR) was the only predictor of elevated baseline MK (p = 0.02). Baseline MK did not correlate with high-sensitivity troponin-I (HsTnI) taken just before CA (p = 0.97), or peak HsTnI during admission (p = 0.74). MK is not a reliable marker of myocardial ischaemia or necrosis. MK increased significantly in all patients following heparin administration in a dose-dependent manner.

Background and objectives: The cytokine midkine (MK) is pathologically implicated in progressive chronic kidney disease (CKD) and its systemic consequences and has potential as both a biomarker and therapeutic target. To date, there are no published data on MK levels in patients with different stages of CKD. This study aims to quantify MK levels in patients with CKD and to identify any correlation with CKD stage, cause, progression, comorbid disease or prescribed medication. Methods: In this observational, single-centre study, demographic data were collected, and serum and urine assayed from 197 patients with CKD and 19 healthy volunteers in an outpatient setting. Results The median serum and urine MK level in volunteers was 754 pg/mL (IQR: 554-1025) and 239 pg/mL (IQR: 154-568), respectively. Compared with serum MK in stage 1 CKD (660 pg/mL, IQR: 417-893), serum MK increased in stage 3 (1878 pg/mL, IQR: 1188-2756; p<0.001), 4 (2768 pg/mL, IQR: 2065-4735; p<0.001) and 5 (4816 pg/mL, IQ: 37477807; p<0.001). Urine MK levels increased from stage 1 CKD (343 pg/mL, IQR: 147-437) to stage 3 (1007 pg/mL, IQR: 465-2766; p=0.07), 4 (2961 pg/mL, IQR: 1368-5686; p=0.005) and 5 (6722 pg/mL, IQR: 3796-10 060; p=0.001). Fractional MK excretion (FeMK) increased from stage 1 CKD (0.159, IQR: 0.145-0.299) to stage 3 (1.024, IQR: 0.451-1.886, p=0.047), 4 (3.39, IQR: 2.10-5.82, p=0.004) and 5 (11.95, IQR: 5.36-24.41, p<0.001). When adjusted for estimated glomerular filtration rate, neither serum nor urine MK correlated with primary CKD diagnosis or CKD progression (small sample). There was a positive correlation between protein:creatinine ratio and FeMK (p=0.003). Angiotensin blockade (adjusted for proteinuria) was associated with lower urine MK (p=0.018) and FeMK (p=0.025). Conclusion MK: levels sequentially rise with CKD stage beyond stage 2, and our data support existing animal evidence for an MK/renin angiotensin-system/proteinuria relationship. To what extent this is related to renal clearance versus pathology, or the consequences of chronically elevated MK levels requires further exploration.

Plants from the family Myoporaceae, which includes the genus Myoporum, are extremely prized by the Australian Aboriginal people for their medicinal properties. Leaves from a plant, which was subsequently identified as Myoporum montanum, were provided for chemical investigation by representatives of an Aboriginal community from the Northern Tablelands district of northern New South Wales, Australia. Acetone extraction of the leaves provided a complex mixture of compounds including sesquiterpene hydrocarbons and more polar furanosesquiterpenes, which were identified by gas-liquid chromatography and retention indices (sesquiterpene hydrocarbons) and spectrometric techniques (furanosesquiterpenes). The major compounds found in a water extract were studied for their antibacterial activity using a disc diffusion assay and for their cell growth inhibition activity. The acetone extract contained sesquiterpene hydrocarbons (~30% of the total extract) in which the major compounds were germacrene-D and bicyclogermacrene. In addition, the extract contained five known toxic furanosesquiterpenes: myoporum ketol, (-)-10,11-dehydroisomyodesmone, (+)-10,11-dehydromyodesmone, 10,11-dehydromyoporum ketol, (-)-10,11-dehydromyoporone, and (±)-myoporone. An aqueous extract of the leaves, emulating the medicinal tea used by the Australian Aboriginal community, was found not to contain significant quantities of the sesquiterpene hydrocarbons and the most toxic furanosesquiterpenes. (±)-Myoporone and (-)-10,11-dehydromyoporone remained in the extract as well as a new furanosesquiterpene, 11-hydroxymyoporone. These three compounds were found to have significant antibacterial activity against Staphylococcus epidermidis, Enterococcus faecalis, and Moraxella catarrhalis but low cytotoxicity against a range of cancer cell lines and normal breast cells at 25µM.

Dysregulation of the inflammatory responses has been suggested to contribute to the events leading to sudden infant deaths. Our objectives were (1) to analyze a single nucleotide polymorphism (SNP) associated with high levels of tumor necrosis factor-a (TNF-a) responses, TNF G-308A, in sudden infant death syndrome (SIDS) infants, SIDS and control parents, and ethnic groups with different incidences of SIDS; (2) the effects of two risk factors for SIDS, cigarette smoke and virus infection, on TNF-a responses; and (3) to assess effects of genotype, cigarette smoke, and gender on TNF-a responses to bacterial toxins identified in SIDS infants. TNF G-308A genotypes were determined by real-time polymerase chain reaction for SIDS infants from Australia, Germany, and Hungary; parents of SIDS infants and their controls; and populations with high (Aboriginal Australian), medium (Caucasian), and low (Bangladeshi) SIDS incidences. Leukocytes from Caucasian donors were stimulated in vitro with endotoxin or toxic shock syndrome toxin-1 (TSST-1). TNF-a responses were measured by L929 bioassay (IU/ml) and assessed in relation to genotype, smoking status, and gender. There was a significantly higher proportion of the minor allele AA genotype among Australian SIDS infants (6/24, 24%) compared to 3/62 (4.8%) controls (p = 0.03). There were no significant differences in TNF-a responses by TNF G-308A genotypes when assessed in relation to smoking status or gender. Given the rarity of the TNF G-308A A allele in Caucasian populations, the finding that 24% of the Australian SIDS infants tested had this genotype requires further investigation and cautious interpretation. Although non-smokers with the AA genotype had higher TNFa responses to both TSST-1 and endotoxin, there were too few subjects with this rare allele to obtain statistically valid results. No effects of genotype, smoking, or gender were observed for TNF-a responses to these toxins.

Previous studies have indicated that cytokine gene polymorphisms of Indigenous Australians were predominantly associated with strong pro-inflammatory responses. We tested the hypothesis that cells of donors with genetic profiles of inflammatory cytokine single nucleotide polymorphisms (SNPs) similar to Indigenous Australians produce higher pro-inflammatory responses. PBMCs from 14 donors with genetic profiles for a high risk of strong pro-inflammatory responses and 14 with low-risk profiles were stimulated with endotoxin and effects of gender, IFN-¿, cigarette smoke extract (CSE) and testosterone on cytokine responses analysed. Cytokines were calculated from standard curves (Luminex 2.3 software). No significant differences were associated with SNP profile alone. Lower pro-inflammatory responses were observed for cells from males with low- or high-risk profiles. For cells from females with high-risk profiles, anti-inflammatory IL-10 responses were significantly reduced. There was no effect of testosterone levels on responses from males. For females, results from IFN-¿-treated cells showed positive correlations between testosterone levels and IL-1ß responses to endotoxin for both risk groups and TNF-a for the high-risk group. If interactions observed among CSE, IFN-¿, genetic background and testosterone reflect those in vivo, these might contribute to increased incidences of hospitalisations for infectious diseases among Indigenous women.

Sudden infant death syndrome (SIDS), neonatal deaths, and deaths from infection are higher among Indigenous Australians. This study aimed to determine the effects of inflammatory responses and exposure to cigarette smoke, two important factors associated with sudden death in infancy, on preterm birth, and birth weight in a cohort of Indigenous mothers. Indigenous Australian women (n = 131) were recruited as part of a longitudinal study while attending antenatal care clinics during pregnancy; blood samples were collected up to three times in pregnancy. Serum cotinine, indicating exposure to cigarette smoke, was detected in 50.4% of mothers. Compared with non-Indigenous women, the cohort had 10 times the prevalence of antibodies to Helicobacter pylori (33 vs. 3%). Levels of immunoglobulin G, antibodies to H. pylori, and C-reactive protein (CRP) were all inversely correlated with gestational age (P < 0.05). CRP levels were positively associated with maternal body mass index (BMI; ¿ = 0.449, P = 0.001). The effects of cigarette smoke (cotinine) and inflammation (CRP) were assessed in relation to risk factors for SIDS: gestational age at delivery and birth weight. Serum cotinine levels were negatively associated with birth weight (¿ = -0.37, P < 0.001), this correlation held true for both male (¿ = -0.39, P = 0.002) and female (¿ = -0.30, P = 0.017) infants. Cotinine was negatively associated with gestational age at delivery (¿ = -0.199, P = 0.023). When assessed by fetal sex, this was significant only for males (¿ = -0.327, P = 0.011). CRP was negatively associated with gestational age at delivery for female infants (¿ = -0.46, P < 0.001). In contrast, maternal BMI was significantly correlated with birth weight. These data highlight the importance of putting programs in place to reduce cigarette smoke exposure in pregnancy and to treat women with chronic infections such as H. pylori to improve pregnancy outcomes and decrease risk factors for sudden death in infancy.

The risk factors for sudden infant death syndrome (SIDS) parallel those associated with susceptibility to or severity of infectious diseases. There is no evidence that a single infectious agent is associated with SIDS; the common thread appears to be induction of inflammatory responses to infections. In this review, interactions between genetic and environmental risk factors for SIDS are assessed in relation to the hypothesis that many infant deaths result from dysregulation of inflammatory responses to "minor" infections. Risk factors are assessed in relation to three important stages of infection: (1) bacterial colonization (frequency or density); (2) induction of temperature-dependent toxins; (3) induction or control of inflammatory responses. In this article, we review the interactions among risk factors for SIDS for their effects on induction or control of inflammatory responses. The risk factors studied are genetic factors (sex, cytokine gene polymorphisms among ethnic groups at high or low risk of SIDS); developmental stage (changes in cortisol and testosterone levels associated with 2- to 4-month age range); environmental factors (virus infection, exposure to cigarette smoke). These interactions help to explain differences in the incidences of SIDS observed between ethnic groups prior to public health campaigns to reduce these infant deaths.

Inflammatory responses have been implicated in several forms of infant deaths (sudden expected deaths and stillbirths) and the initiation of pre-term births. In this study, we examined matched samples of term maternal blood, cord blood, and amniotic fluid obtained from 24 elective cesarean deliveries for both pro- and anti-inflammatory cytokines thought to be important in maintaining a balanced response leading to successful pregnancy outcome. These included interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-a (TNF-a), interferon-¿ (IFN-¿), IL-10, and IL-1 receptor antagonist (IL-1ra). Amniotic fluid levels for each of the cytokines examined were significantly higher than those for cord blood or maternal plasma. While pro-inflammatory cytokines were higher in amniotic fluid associated with male fetuses compared with females, the major significant difference was higher levels of IL-1ra in amniotic fluid associated with female fetuses. Our study supports similar findings for cytokines during mid-trimester, which noted that amniotic fluid levels were higher than those in maternal blood. Our study suggests that maternal decidua secretes additional IL-ra in the presence of a female conceptus which improves the likelihood of a good outcome compared to pregnancies with male fetuses.

Respiratory infections have been implicated in sudden infant death syndrome (SIDS). As interferon-¿ (IFN-¿) is a major response to virus infection, we examined (1) the frequency of single nucleotide polymorphism (SNP), IFNG T + 874A, in SIDS infants, their parents, and ethnic groups with different incidences of SIDS; (2) model systems with a monocytic cell line (THP-1) and human peripheral blood monocytes (PBMC) for effects of levels of IFN-¿ on inflammatory responses to bacterial antigens identified in SIDS; (3) interactions between genetic and environmental factors on IFN-¿ responses. IFNG T + 874A genotypes were determined for SIDS infants from three countries; families who had a SIDS death; populations with high (Indigenous Australian), medium (Caucasian), and low (Bangladeshi) SIDS incidences. The effect of IFN-¿ on cytokine responses to endotoxin was examined in model systems with THP-1 cells and human PBMC. The IFN-¿ responses to endotoxin and toxic shock syndrome toxin (TSST-1) were assessed in relation to genotype, gender, and reported smoking. There was a marginal association with IFNG T + 874A genotype and SIDS (p = 0.06). Indigenous Australians had significantly higher proportions of the IFNG T + 874A SNP (TT) associated with high responses of IFN-¿. THP-1 cells showed a dose dependent effect of IFN-¿ on cytokine responses to endotoxin. For PBMC, IFN-¿ enhanced interleukin (IL)-1ß, IL-6, and tumor necrosis factor-a responses but reduced IL-8 and IL-10 responses. Active smoking had a suppressive effect on baseline levels of IFN-¿. There was no effect of gender or genotype on IFN-¿ responses to bacterial antigens tested; however, significant differences were observed between genotypes in relation to smoking. The results indicate virus infections contribute to dysregulation of cytokine responses to bacterial antigens and studies on physiological effects of genetic factors must include controls for recent or concurrent infection and exposure to cigarette smoke.

Interactions among major risk factors associated with bacterial infections were assessed in a model system using surrogates for virus infection; IFN-g, and exposure to cigarette smoke; cigarette smoke extract (CSE), nicotine and cotinine. Cytokine responses elicited by LPS from THP-1 cells in the presence of these components, or combinations of components, were assessed by multiplex bead assay, i.e. IL-1ß, IL-6, IL-8, IL-10, TNF-a and IFN-¿. IFN-¿-priming significantly increased pro-inflammatory cytokines induced by LPS. CSE suppressed production of pro-inflammatory cytokines IL-1ß, TNF-a and IFN-¿, but enhanced production of IL-8. Nicotine and cotinine suppressed all cytokine responses. In combination, IFN-¿ masked the inhibitory effects of CSE. In relation to the objectives of the study, we concluded that (a) IFN¿ at biologically relevant concentrations significantly enhanced pro-inflammatory responses; (b) CSE, nicotine and cotinine dysregulated the inflammatory response and that the effects of CSE were different from those of the individual components, nicotine and cotinine; (c) when both IFN-¿ and CSE were present, IFN-¿ masked the effect of CSE. There is a need for clinical investigations on the increase in IL-8 responses in relation to exposure to cigarette smoke and increased pro-inflammatory responses in relation to recent viral infection. © 2013 The Author(s).