Translocation of Antimicrobial Peptides across Model Membranes: The Role of Peptide Chain Length.

Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 (24Hst5), focusing on the effect of the length of the peptide chain. 24Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter (14Hst5) and a longer (48Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24Hst5. On the contrary, a cushion is formed both in the case of 14Hst5 and 48Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.

Interaction of a Histidine-Rich Antimicrobial Saliva Peptide with Model Cell Membranes: The Role of Histidines.

Histatin 5 is a histidine-rich, intrinsically disordered, multifunctional saliva protein known to act as a first line of defense against oral candidiasis caused by Candida albicans. An earlier study showed that, upon interaction with a common model bilayer, a protein cushion spontaneously forms underneath the bilayer. Our hypothesis is that this effect is of electrostatic origin and that the observed behavior is due to proton charge fluctuations of the histidines, promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Here we are investigating the role of the histidines in more detail by defining a library of variants of the peptide, where the former have been replaced by the pH-insensitive amino acid glutamine. By using experimental techniques such as circular dichroism, small angle X-ray scattering, quartz crystal microbalance with dissipation monitoring, and neutron reflectometry, it was determined that changing the number of histidines in the peptide sequence did not affect the structure of the peptide dissolved in solution. However, it was shown to affect the penetration depth of the peptide into the bilayer, where all variants except the one with zero histidines were found below the bilayer. A decrease in the number of histidine from the original seven to zero decreases the ability of the peptide to penetrate the bilayer, and the peptide is then also found residing within the bilayer. We hypothesize that this is due to the ability of the histidines to charge titrate, which charges up the peptide, and enables it to penetrate and translocate through the lipid bilayer.

Spontaneous Formation of Cushioned Model Membranes Promoted by an Intrinsically Disordered Protein.

In this article, it is shown that by exposing commonly used lipids for biomembrane mimicking studies, to a solution containing the histidine-rich intrinsically disordered protein histatin 5, a protein cushion spontaneously forms underneath the bilayer. The underlying mechanism is attributed to have an electrostatic origin, and it is hypothesized that the observed behavior is due to proton charge fluctuations promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Hence, we anticipate that this novel "green" approach of forming cushioned bilayers can be an important tool to mimic the cell membrane without the disturbance of the solid substrate, thereby achieving a further understanding of protein-cell interactions.

Comorbidity and long-term outcome in patients with congenital heart block and their siblings exposed to Ro/SSA autoantibodies in utero.

OBJECTIVE: Congenital heart block (CHB) may develop in fetuses of Ro/SSA autoantibody-positive women. Given the rarity of CHB, information on comorbidity and complications later in life is difficult to systematically collect for large groups of patients. We therefore used nation-wide healthcare registers to investigate comorbidity and outcomes in patients with CHB and their siblings. METHODS: Data from patients with CHB (n= 119) and their siblings (n= 128), all born to anti-Ro/SSA-positive mothers, and from matched healthy controls (n= 1,190) and their siblings (n= 1,071), were retrieved from the Swedish National Patient Register. Analyses were performed by Cox proportional hazard modelling. RESULTS: Individuals with CHB had a significantly increased risk of cardiovascular comorbidity, with cardiomyopathy and/or heart failure observed in 20 (16.8%) patients versus 3 (0.3%) controls, yielding a HR of 70.0 (95% CI 20.8 to 235.4), and with a HR for cerebral infarction of 39.9 (95% CI 4.5 to 357.3). Patients with CHB also had a higher risk of infections. Pacemaker treatment was associated with a decreased risk of cerebral infarction but increased risks of cardiomyopathy/heart failure and infection. The risk of systemic connective tissue disorder was also increased in patients with CHB (HR 11.8, 95% CI 4.0 to 11.8), and both patients with CHB and their siblings had an increased risk to develop any of 15 common autoimmune conditions (HR 5.7, 95% CI 2.83 to 11.69 and 3.6, 95% CI 1.7 to 8.0, respectively). CONCLUSIONS: The data indicate an increased risk of several cardiovascular, infectious and autoimmune diseases in patients with CHB, with the latter risk shared by their siblings.

Outcome in 212 anti-Ro/SSA-positive pregnancies and population-based incidence of congenital heart block.

INTRODUCTION: We investigated the effects of maternal autoimmune disease and fetal congenital heart block (CHB) on pregnancy outcomes in anti-Ro/SSA-positive women and assessed the population-based incidence of isolated CHB. MATERIAL AND METHODS: One hundred and ninety nine anti-Ro/SSA-positive pregnancies were prospectively followed at our center (2000-2013). Seven fetuses developed atrioventricular block (AVB) II-III. In this period, another 13 anti-Ro/SSA-positive pregnancies were referred for fetal bradycardia, subsequently diagnosed with AVB II-III. Cesarean section rates, gestational age, body measurements at birth, and the incidence of CHB in these 212 pregnancies were analyzed in relation to fetal atrioventricular conduction and maternal diagnosis and compared with data from the Medical Birth Registry on 352,104 pregnancies in the Stockholm County. RESULTS: The prevalence of maternal systemic lupus erythematosus (SLE) and primary Sjögren's syndrome and the outcomes at birth were similar in normal conduction and AVB I cases. Only 1/20 AVB II-III cases (0/7 in the surveillance group) had a mother diagnosed with SLE, compared with 73/192 in cases with normal conduction or AVB I. Excluding cases with AVB II-III, SLE mothers more frequently delivered by cesarean section (31% vs. 20%, p < 0.05) and had a higher incidence of preterm birth (13% vs. 5.8%, p < 0.05) than the county population. Both SLE and primary Sjögren's syndrome mothers had a fourfold greater rate of growth-retarded babies (10.11% vs. 2.2%, p < 0.001). The incidence of autoantibody-related AVB II-III in Stockholm County was 1/23 300. CONCLUSION: This study of CHB provides new information on the incidence of CHB and outcome of pregnancy in anti-Ro/SSA-positive women, which has clinical relevance when counseling rheumatic patients considering pregnancy.

Late development of complete atrioventricular block may be immune mediated and congenital in origin.

AIM: To investigate the correlation between maternal autoantibodies and age at diagnosis of isolated complete atrioventricular (AV) block (CAVB) and to study signs of late progression of foetal immune-mediated insults in cases of postnatally diagnosed CAVB. METHODS: Patients with CAVB (n = 190) identified in a population-based manner were included. Maternal autoantibody profile was correlated with age at CAVB diagnosis. A structured review of medical records was performed if a late CAVB diagnosis (>27 days post-partum) was associated with a sero-positive mother. RESULTS: Maternal Ro/La autoantibodies were observed in 88% of cases with a congenital diagnosis. Thirteen cases with a sero-positive mother and late CAVB diagnosis were found (age-range: 4 months-43 years). In two cases, CAVB was diagnosed in conjunction with infections, one case had a family history of cardiomyopathy and two cases had nontypical clinical presentations, indicating alternative pathogenetic mechanisms. In the remaining eight cases, no likely factors inducing CAVB, other than maternal autoantibodies, could be identified. CONCLUSION: Our observations support the hypothesis that late progression to CAVB can be the result of an immune-mediated pathogenetic mechanism during foetal life. An autoantibody-associated diagnosis after the neonatal period is therefore possible, and testing of maternal serology at the time of diagnosis is recommended.

Neurodevelopment in children with and without congenital heart block born to anti-Ro/SSA-positive mothers.

OBJECTIVE: To define factors influencing neurodevelopment in children with and without complete congenital heart block (CHB) born to mothers with Ro/SSA autoantibodies. PATIENTS AND METHODS: Medical records of a population-based cohort of siblings with (n = 60) and without (n = 54) CHB born 1974-2009 to anti-Ro/SSA-positive mothers were retrieved from children primary healthcare centres and school health services and used to extract data on neurodevelopment. RESULTS: Impaired neurodevelopment was reported in 16% of the children (18/114) during the follow-up time of 13.0 (8.2-17.5) years, median (quartiles). Reported problems included speech (9%), motor (8%) and learning (8%) impairment, attention deficit (5%) and behavioural impairment (4%). Impairment in motor skill development was more common in boys (p < 0.001) if the child was born preterm (p < 0.001). Learning impairment was significantly influenced by maternal SLE (p < 0.005), while attention deficits was influenced by both maternal SLE (p < 0.05) and CHB in the child (p < 0.05). CONCLUSIONS: Our data indicate that in addition to well-established factors such as male sex and being born preterm, both maternal SLE and CHB may influence neurodevelopment. Follow-up of neurodevelopment should therefore be considered for children with CHB, especially if the mother is diagnosed with SLE.

Long-term growth of children with autoantibody-mediated congenital heart block.

AIM: To analyse growth of children with and without congenital heart block (CHB) born to anti-Ro/SSA positive mothers from birth to 18 years of age, using a population-based cohort of Swedish CHB patients. METHODS: Medical records for siblings with (n = 72) and without (n = 60) CHB born 1973-2009 to anti-Ro/SSA positive mothers were retrieved from child healthcare centres and school health services and used to extract data on growth from birth to 18 years. RESULTS: Compared with reference standards, children with CHB were retarded in weight by 0.75-1.0 SD from birth to 2-3 years of age. Thereafter, the CHB children started to catch up, reaching the reference standards at 9-11 years of age. Pacemaker treatment was not correlated with the catch-up in growth. Individuals with CHB were retarded in both weight and height from birth to 9-11 years of age when compared to siblings without CHB, who did not demonstrate restriction in these measurements. CONCLUSION: Presence of CHB is a more important predictor of growth restriction than maternal rheumatic disease and foetal anti-Ro/SSA exposure. The restriction persists for several years after birth, despite pacemaker treatment, which highlights the importance of follow-up of children with CHB regarding nutrition and growth.

Development of heart block in children of SSA/SSB-autoantibody-positive women is associated with maternal age and displays a season-of-birth pattern.

OBJECTIVE: Congenital heart block may develop in the fetuses of Ro/SSA-positive and La/SSB-positive mothers. Recurrence rates of only 10-20% despite persisting maternal antibodies indicate that additional factors are critical for the establishment of heart block. The authors investigated the influence of other maternal and fetal factors on heart block development in a Swedish population-based cohort. METHODS: The influence of fetal gender, maternal age, parity and time of birth on heart block development was analysed in 145 families, including Ro/La-positive (n=190) and Ro/La-negative (n=165) pregnancies. RESULTS: There was a recurrence rate of 12.1% in Ro/La-positive women, and no recurrence in Ro/La-negative women. Fetal gender and parity did not influence the development of heart block in either group. Maternal age in Ro/La-positive pregnancies with a child affected by heart block was, however, significantly higher than in pregnancies resulting in babies without heart block (p<0.05).Seasonal timing of pregnancy influenced the outcome. Gestational susceptibility weeks 18-24 occurring during January-March correlated with a higher proportion of children with heart block and lower vitamin D levels during the same period in a representative sample of Swedish women and a corresponding higher proportion of children with heart block born in the summer (p<0.02). Maternal age or seasonal timing of pregnancy did not affect the outcome in Ro/La-negative pregnancies. CONCLUSION: This study identifies maternal age and seasonal timing of pregnancy as novel risk factors for heart block development in children of Ro/La-positive women. These observations may be useful for counselling when pregnancy is considered.

Outcome in young patients with isolated complete atrioventricular block and permanent pacemaker treatment: A nationwide study of 127 patients.

BACKGROUND: Subgroups of pacemaker (PM)-treated children with isolated complete atrioventricular block are at risk of developing left ventricular (LV) dysfunction. OBJECTIVES: We aimed to compare the long-term outcome in anti-SSA-Ro/SSB-La antibody-exposed (AB+) and unexposed (AB-) patients and identify preimplantation variables associated with poor outcome. METHODS: In total, 127 PM-treated patients aged 0-17 years with isolated complete atrioventricular block were studied retrospectively. RESULTS: Sixty-three patients were diagnosed prenatally, of whom 92% were AB+. Before PM treatment, fractional shortening (FS) z-score was significantly lower in AB+ patients than in AB- patients (-0.14 ± 3.6 vs 2.03 ± 2.3). Before PM implantation, there were sex differences (male/female) in median time from diagnosis to PM implantation (0.2 years vs 1.0 years), median neonatal heart rate (50 beats/min vs 60 beats/min), left ventricular end-diastolic dimension (LVEDD) z-score (2.68 ± 1.41 vs 1.74 ± 1.40), and FS (-0.19 ± 3.38 vs 1.42 ± 2.98). The median age at PM implantation was 3.2 years, and median follow-up was 8.7 years. At follow-up, LVEDD and FS did not differ significantly between AB+ and AB- patients, but LVEDD was higher in patients diagnosed before 1 month of age. Nine patients (8%) developed LV dysfunction, and 4 died. LV dysfunction was associated with diagnosis before 1 month of age and abnormal LV function before PM implantation. CONCLUSION: LV dysfunction at follow-up was seen only in cases diagnosed before 1 month, with abnormal echocardiography before PM implantation. Boys had a more compromised cardiac status and were paced at an earlier age than girls. Fetal AB exposure and male sex were related to abnormal LV function before PM therapy, but not at follow-up.

Outcome and growth of infants fetally exposed to heart block-associated maternal anti-Ro52/SSA autoantibodies.

OBJECTIVE: The purpose of this work was to analyze outcome with focus on growth in infants fetally exposed to heart block-associated maternal anti-Ro52/SSA autoantibodies and identify maternal factors other than the autoantibodies increasing the risk of fetal heart block. PATIENTS AND METHODS: Thirty-two pregnancies in 30 anti-Ro52-positive mothers were included. Seven fetuses developed second-degree or third-degree atrioventricular block, 8 developed first-degree atrioventricular block, and 17 had normal atrioventricular conduction, as diagnosed by using Doppler echocardiography. Three of 6 surviving fetuses with second-degree or third-degree atrioventricular block received treatment with fluorinated steroids. Two fetuses with second-degree atrioventricular block converted to first-degree atrioventricular block without any signs of progression during the study period. Maternal and longitudinal infant data were collected from planned neonatal follow-up and childhood health records from birth to 12 months of age in 31 survivors. RESULTS: Women giving birth to infants with prenatal second-degree or third-degree atrioventricular block were older and with higher parity than those with first-degree atrioventricular block or normal atrioventricular conduction. Second-degree or third-degree atrioventricular block pregnancies were <40 completed weeks, whereas pregnancies with first-degree atrioventricular block or normal atrioventricular conduction had a normal duration. Fetuses with second-degree or third-degree atrioventricular block were retarded by -0.98 +/- 0.77 SD in weight at birth and did not show any catch-up during infancy. In contrast, fetuses with first-degree atrioventricular block or normal atrioventricular conduction had a weight reduction of -0.51 +/- 1.01 SD with a catch-up during the first months after birth. CONCLUSIONS: This report documents that newborns with autoantibody-mediated second-degree or third-degree atrioventricular block are retarded in growth, with no catch-up during infancy, whereas fetuses with first-degree atrioventricular block or normal atrioventricular conduction have a normal growth soon after birth. Increased maternal age and/or parity seem to carry an increased risk for fetal heart block.