Toll-Like Receptor 9 Mediated Responses in Cardiac Fibroblasts

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and –C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.     

MHC Class II Molecules Enhance Toll-Like Receptor Mediated Innate Immune Responses

Background

Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.

Methodology/Principal Findings

We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-β-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.

Conclusions/Significance

These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.     

Acquisition of Adult-Like TLR4 and TLR9 Responses during the First Year of Life

Background

Characteristics of the human neonatal immune system are thought to be responsible for heightened susceptibility to infectious pathogens and poor responses to vaccine antigens. Using cord blood as a source of immune cells, many reports indicate that the response of neonatal monocytes and dendritic cells (DC) to Toll-like receptor (TLR) agonists differs significantly from that of adult cells. Herein, we analyzed the evolution of these responses within the first year of life.

Methodology/Principal Findings

Blood samples from children (0, 3, 6, 9, 12 month old) and healthy adults were stimulated ex vivo with bacterial lipopolysaccharide (LPS, TLR4 agonist) or CpG oligonucleotides (TLR9 agonist). We determined phenotypic maturation of monocytes, myeloid (m) and plasmacytoid (p) DC and production of cytokines in the culture supernatants. We observed that surface expression of CD80 and HLA-DR reaches adult levels within the first 3 months of life for mDCs and 6–9 months of life for monocytes and pDCs. In response to LPS, production of TNF-α, IP-10 and IL-12p70 reached adult levels between 6–9 months of life. In response to CpG stimulation, production of type I IFN-dependent chemokines (IP-10 and CXCL9) gradually increased with age but was still limited in 1-year old infants as compared to adult controls. Finally, cord blood samples stimulated with CpG ODN produced large amounts of IL-6, IL-8, IL-1β and IL-10, a situation that was not observed for 3 month-old infants.

Conclusions

The first year of life represents a critical period during which adult-like levels of TLR responses are reached for most but not all cytokine responses.     

Functional Connectivity in the First Year of Life in Infants at Risk for Autism Spectrum Disorder: An EEG Study

In the field of autism research, recent work has been devoted to studying both behavioral and neural markers that may aide in early identification of autism spectrum disorder (ASD). These studies have often tested infants who have a significant family history of autism spectrum disorder, given the increased prevalence observed among such infants. In the present study we tested infants at high- and low-risk for ASD (based on having an older sibling diagnosed with the disorder or not) at 6- and 12-months-of-age. We computed intrahemispheric linear coherence between anterior and posterior sites as a measure of neural functional connectivity derived from electroencephalography while the infants were listening to speech sounds. We found that by 12-months-of-age infants at risk for ASD showed reduced functional connectivity compared to low risk infants. Moreover, by 12-months-of-age infants later diagnosed with ASD showed reduced functional connectivity, compared to both infants at low risk for the disorder and infants at high risk who were not later diagnosed with ASD. Significant differences in functional connectivity were also found between low-risk infants and high-risk infants who did not go onto develop ASD. These results demonstrate that reduced functional connectivity appears to be related to genetic vulnerability for ASD. Moreover, they provide further evidence that ASD is broadly characterized by differences in neural integration that emerge during the first year of life.     

Association of Cytokine and Toll-Like Receptor Gene Polymorphisms with Severe Malaria in Three Regions of Cameroon

P. falciparum malaria is one of the most widespread and deadliest infectious diseases in children under five years in endemic areas. The disease has been a strong force for evolutionary selection in the human genome, and uncovering the critical human genetic factors that confer resistance to the disease would provide clues to the molecular basis of protective immunity that would be invaluable for vaccine development. We investigated the effect of single nucleotide polymorphisms (SNPs) on malaria pathology in a case- control study of 1862 individuals from two major ethnic groups in three regions with intense perennial P. falciparum transmission in Cameroon. Twenty nine polymorphisms in cytokine and toll-like receptor (TLR) genes as well as the sickle cell trait (HbS) were assayed on the Sequenom iPLEX platform. Our results confirm the known protective effect of HbS against severe malaria and also reveal a protective effect of SNPs in interleukin-10 (IL10) cerebral malaria and hyperpyrexia. Furthermore, IL17RE rs708567 GA and hHbS rs334 AT individuals were associated with protection from uncomplicated malaria and anaemia respectively in this study. Meanwhile, individuals with the hHbS rs334 TT, IL10 rs3024500 AA, and IL17RD rs6780995 GA genotypes were more susceptible to severe malarial anaemia, cerebral malaria, and hyperpyrexia respectively. Taken together, our results suggest that polymorphisms in some immune response genes may have important implications for the susceptibility to severe malaria in Cameroonians. Moreover using uncomplicated malaria may allow us to identify novel pathways in the early development of the disease.     

Accuracy of Two Motor Assessments during the First Year of Life in Preterm Infants for Predicting Motor Outcome at Preschool Age

Aim

The primary aim of this study was to investigate the accuracy of the Alberta Infant Motor Scale (AIMS) and Neuro-Sensory Motor Developmental Assessment (NSMDA) over the first year of life for predicting motor impairment at 4 years in preterm children. The secondary aims were to assess the predictive value of serial assessments over the first year and when using a combination of these two assessment tools in follow-up.

Method

Children born <30 weeks’ gestation were prospectively recruited and assessed at 4, 8 and 12 months’ corrected age using the AIMS and NSMDA. At 4 years’ corrected age children were assessed for cerebral palsy (CP) and motor impairment using the Movement Assessment Battery for Children 2^nd-edition (MABC-2). We calculated accuracy of the AIMS and NSMDA for predicting CP and MABC-2 scores ≤15^th (at-risk of motor difficulty) and ≤5^th centile (significant motor difficulty) for each test (AIMS and NSMDA) at 4, 8 and 12 months, for delay on one, two or all three of the time points over the first year, and finally for delay on both tests at each time point.

Results

Accuracy for predicting motor impairment was good for each test at each age, although false positives were common. Motor impairment on the MABC-2 (scores ≤5^th and ≤15^th) was most accurately predicted by the AIMS at 4 months, whereas CP was most accurately predicted by the NSMDA at 12 months. In regards to serial assessments, the likelihood ratio for motor impairment increased with the number of delayed assessments. When combining both the NSMDA and AIMS the best accuracy was achieved at 4 months, although results were similar at 8 and 12 months.

Interpretation

Motor development during the first year of life in preterm infants assessed with the AIMS and NSMDA is predictive of later motor impairment at preschool age. However, false positives are common and therefore it is beneficial to follow-up children at high risk of motor impairment at more than one time point, or to use a combination of assessment tools.

Trial Registration

ACTR.org.au ACTRN12606000252516     

Growth of HIV-Exposed Uninfected Infants in the First 6 Months of Life in South Africa: The IeDEA-SA Collaboration

BackgroundHIV-exposed uninfected (HEU) infants are a growing population in sub-Saharan Africa especially with the increasing coverage of more effective prevention of mother-to-child transmission (PMTCT) antiretroviral therapy regimens. This study describes the characteristics of South African HEU infants, investigates factors impacting birth weight and assesses their growth within the first 28 weeks of life.MethodsThis is a retrospective cohort based on routine clinical data from two South African PMTCT programmes. Data were collected between 2007 and 2013. Linear regression assessed factors affecting birth weight-for-age z-scores (WAZ) while growth (longitudinal WAZ) was assessed using mixed effects models.ResultsWe assessed the growth of 2621 HEU infants (median birth WAZ was -0.65 (IQR -1.46; 0.0) and 51% were male). The feeding modalities practised were as follows: 0.5% exclusive breastfeeding, 7.9% breastfeeding with unknown exclusivity, 0.08% mixed breastfeeding and 89.2% formula feeding. Mothers with CD4 <200 cells/μl delivered infants with a lower birth WAZ (adjusted ß -0.253 [95% CI -0.043; -0.072], p = 0.006) compared to mothers with aCD4 ≥500 cells/μl. Similarly, mothers who did not receive antiretroviral drugs delivered infants with a lower birth WAZ (adjusted ß -0.39 [95% CI -0.67; -0.11], p = 0.007) compared to mothers who received antenatal antiretrovirals. Infants with a birth weight <2 500g (ß 0.070 [95% CI 0.061; 0.078], p <0.0001) experienced faster growth within the first 28 weeks of life compared to infants with a birth weight ≥2 500g. Infants with any breastfeeding exposure experienced slower longitudinal growth compared to formula fed infants (adjusted ß -0.012 [95% CI 0.021; -0.003], p = 0.011).ConclusionLess severe maternal disease and the use of antiretrovirals positively impacts birth weight in this cohort of South African HEU infants. Formula feeding was common with breastfed infants experiencing marginally slower longitudinal growth.     

Evaluation of Toll-Like Receptor 2 and 4 RNA Expression and the Cytokine Profile in Postmenopausal Women with Metabolic Syndrome

Objective

To evaluate the gene expression of Toll-Like (TLR-2 and TLR-4) receptors and cytokine profile in postmenopausal women with or without metabolic syndrome (MetS).

Methods

In this cross-sectional study, 311 Brazilian women (age≥45 years and amenorrhea≥12 months) were included. Women showing three or more of the following diagnostic criteria were diagnosed as positive for MetS: waist circumference>88 cm, triglycerides≥150 mg/dL, HDL cholesterol<50 mg/dL, blood pressure≥130/85 mmHg, and fasting glucose≥100 mg/dL. The expression of TLR-2 and TLR-4 in peripheral blood was evaluated by RNA extraction and subsequent real time PCR analysis. The cytokine profile, tumor necrosis factor alpha (TNF-α) and interleukins 1β, 6, and 10, were measured by ELISA.

Results

The expression of TLR-2 RNA was demonstrated in 32.5% and TLR-4 in 20.6% of the subjects. There was no association between the expression of TLR-2 and TLR-4 and the presence or absence of MetS (P>0.05). A greater production of IL-6 was associated with TLR-2 and TLR-4 expressions and greater production of TNF-α was associated only with TLR-2 expression (P>0.05). Only the lower quartile of IL-10 was associated with the presence of the MetS (P>0.05).

Conclusions

TLR-2 and TLR-4 expressions were associated with increased pro-inflammatory cytokines, IL-6 and TNF-α, with no association with biomarkers of MetS. The low concentrations of IL-10 may suggest an anti-inflammatory modulation in postmenopausal women with MetS.     

Host Avian Beta-Defensin and Toll-Like Receptor Responses of Pigeons following Infection with Pigeon Paramyxovirus Type 1

The high morbidity and mortality in pigeons caused by pigeon paramyxovirus type 1 (PPMV-1) highlights the need for new insights into the host immune response and novel treatment approaches. Host defense peptides (HDPs) are key components of the innate immune system. In this study, three novel avian β-defensins (AvBDs 2, 7, and 10) were characterized in pigeons and shown to possess direct antiviral activity against PPMV-1 in vitro. In addition, we evaluated the mRNA expression of these AvBDs and other immune-related genes in tissues of 2-month-old infected pigeons at 3 and 7 days postinfection. We observed that the expression of AvBD2 in the cecal tonsil, lungs, and proventriculus, as well as the expression of AvBD10 in the spleen, lungs, proventriculus, and kidneys, was upregulated in infected pigeons. Similarly, the expression of both Toll-like receptor 3 (TLR3) and TLR7 was increased in the spleen, trachea, and proventriculus, while TLR15 expression was increased only in the lungs of infected pigeons. In addition, inducible nitric oxide synthase (iNOS) expression was upregulated in the spleen, the bursa of Fabricius, the trachea, and the proventriculus of infected pigeons. Furthermore, we observed a high correlation between the expression of AvBD2 and the expression of either TLR7 or TLR15, as well as between AvBD10 expression and either TLR3 or TLR7 expression in respective tissues. The results suggest that PPMV-1 infection can induce innate host responses characterized by the activation of TLRs, particularly TLR3 and TLR7, AvBDs (2 and 10), and iNOS in pigeons.     

Changes in the Relative Inflammatory Responses in Sheep Cells Overexpressing of Toll-Like Receptor 4 When Stimulated with LPS

Background

Many groups of Gram-negative bacteria cause diseases harmful to sheep. TLR4 is an important Toll-like receptor (TLR) which responds to common Gram-negative bacterial infections. Activation of TLR4 leads to the induction of inflammatory responses, which is a linkage between the innate and adaptive immune systems. A vector pTLR4-3S was constructed to overexpress TLR4 gene in sheep. In this study, effects of TLR4 overexpression on inflammation response under LPS stimulated were addressed in vivo and in vitro.

Methodology/Principal Findings

Sheep fetal fibroblasts were transfected with expression vector pTLR4-3S. Transgenic sheep were produced by microinjection of the constructed plasmids into fertilized eggs. Fetal fibroblasts, monocyte-macrophage and fibroblasts isolated from the transgenic sheep were stimulated by LPS. After that immunoactive factors (TNF-α, IL-10, IL-6, IL-8, IFN-γ), nitric oxide, phagocytize ability and adhesion were detected. Furthermore, transgenic sheep were intradermal injected of LPS in ear and observed pathological changes by HE strain. Overexpression of TLR4 gene was observed on transgenic cells and individuals. In vitro, TLR4 overexpression transgenic cells secreted Th1 and Th2 inducing cytokines with a strong LPS mediated inflammation response and promoting the secretion of nitric oxide, and then recovered to initial level. The phagocytosis index of monocyte/macrophage in transgenic sheep was higher than that of non-transgenic sheep (P<0.05). In vivo, tissue sections showed that transgenic individuals launched inflammation response more quickly.

Conclusions/Significance

Overexpression of TLR4 in transgenic sheep enhanced the clearance of invaded microbe through secretion of cytokines, activation of macrophage, oxidation damage and infiltration of neutrophil.     

The Faces in Infant-Perspective Scenes Change over the First Year of Life

Mature face perception has its origins in the face experiences of infants. However, little is known about the basic statistics of faces in early visual environments. We used head cameras to capture and analyze over 72,000 infant-perspective scenes from 22 infants aged 1-11 months as they engaged in daily activities. The frequency of faces in these scenes declined markedly with age: for the youngest infants, faces were present 15 minutes in every waking hour but only 5 minutes for the oldest infants. In general, the available faces were well characterized by three properties: (1) they belonged to relatively few individuals; (2) they were close and visually large; and (3) they presented views showing both eyes. These three properties most strongly characterized the face corpora of our youngest infants and constitute environmental constraints on the early development of the visual system.     

Prospective Study of Serum Cholesterol Levels during First Year of Life

A longitudinal prospective study of serum cholesterol concentrations during the first year of life has been carried out in 302 healthy babies. The results show that serum cholesterol estimations in cord blood cannot be used as a screening test for the diagnosis of familial hypercholesterolaemia. The only child subsequently found to have the condition had a cord serum cholesterol of 85 mg/100 ml compared with the mean value for the group of 78 mg/100 ml. The babies who had cord values greater than 100 mg/100 ml had values distributed throughout the normal range when re-examined at 1 year of age. Serum cholesterol concentrations during the early months of life were markedly influenced by the type of milk fed; it is suggested that investigations to establish the diagnosis of familial hypercholesterolaemia are deferred until the child is about 1 year old and feeding with cows'' milk and mixed diet is established.Values obtained for serum cholesterol concentrations (mg/100 ml, mean ± 1 S.D.) in healthy infants in this study were: at birth 78 ± 23, at 1 week 155 ± 31, at 6 weeks 155 ± 31, at 4 months 184 ± 36, at 8 months 195 ± 37, and at 1 year 191 ± 36.     

Ontogeny and evolution of the flowers of South African Restionaceae with special emphasis on the gynoecium

 The South African Restionaceae make up a highly diverse group of genera displaying several reductive trends in the configuration of the flower, especially in the gynoecium. In this paper the floral ontogeny of fourteen species representing nine of the 11 genera of the Restio clade is studied with the SEM. Although flowers are basically simple, the variability in both mature and developmental stages is striking. Differences between species are the result of changes in growth rate, coupled with differential pressures of organs. Trends in the elaboration of bracts, perianth, androecium and gynoecium are compared. Together with data that have been presented elsewhere about the other clade of African Restionaceae, viz. the Willdenowia-clade, a scheme with potential developmental pathways is proposed and the most evident routes are selected based on ontogenetic evidence. Nine possible reductions are presented arising through three main routes. Received August 27, 2001 Accepted October 26, 2001     

Leprosy and the Adaptation of Human Toll-Like Receptor 1

Leprosy is an infectious disease caused by the obligate intracellular pathogen Mycobacterium leprae and remains endemic in many parts of the world. Despite several major studies on susceptibility to leprosy, few genomic loci have been replicated independently. We have conducted an association analysis of more than 1,500 individuals from different case-control and family studies, and observed consistent associations between genetic variants in both TLR1 and the HLA-DRB1/DQA1 regions with susceptibility to leprosy (TLR1 I602S, case-control P = 5.7×10⁻⁸, OR = 0.31, 95% CI = 0.20–0.48, and HLA-DQA1 rs1071630, case-control P = 4.9×10⁻¹⁴, OR = 0.43, 95% CI = 0.35–0.54). The effect sizes of these associations suggest that TLR1 and HLA-DRB1/DQA1 are major susceptibility genes in susceptibility to leprosy. Further population differentiation analysis shows that the TLR1 locus is extremely differentiated. The protective dysfunctional 602S allele is rare in Africa but expands to become the dominant allele among individuals of European descent. This supports the hypothesis that this locus may be under selection from mycobacteria or other pathogens that are recognized by TLR1 and its co-receptors. These observations provide insight into the long standing host-pathogen relationship between human and mycobacteria and highlight the key role of the TLR pathway in infectious diseases.     

Fate of Surviving Low-birth-weight Infants with Coagulation Deficiencies on the First Day of Life

Fifty-two surviving low-birth-weight infants who had low Thrombotest (Owren) results on the first day, together with the same number of matched controls with higher Thrombotest results, were examined for the integrity of their central nervous system. Gross abnormalities were found in 13·5% of the low Thrombotest group compared with 1·9% in the higher group. Minor brain damage syndromes were more common in the low Thrombotest group. The combined brain damage syndromes were 23·1% in the low Thrombotest group compared with 3·8% in the higher group.It is suggested that the causes of the brain damage in the low Thrombotest group are either non-fatal cerebral haemorrhage or intravascular fibrin deposition associated with disseminated intravascular coagulation.     

The Word as Conditioned Inhibitor in Infants During the First Three Years of Life

The purpose of this experiment was to compare the features of the development of conditioned inhibition to verbal and non-verbal stimuli in early childhood.     

Prognostic Implications of Electroencephalographic Findings of Hypsarrhythmia in First Year of Life

One hundred and five children who had shown the electroencephalographic (E.E.G.) features of hypsarrhythmia in the first year of life (1956-62) were assessed in 1969 in respect of death and mental development. Irrespective of the presenting symptomatology, and to some extent irrespective of therapy, mortality in the group was of the order of one in four (mostly before the age of 3 years) and the incidence of mental subnormality in the survivors was 77%. Only 18 children attained fairly normal standards of mental development and could attend ordinary schools. It seems that whatever the clinical picture at the time, the presence of hypsarrhythmia in the E.E.G. of an infant is a physical sign of grave prognostic implications.     

Soluble Markers of the Toll-Like Receptor 4 Pathway Differentiate between Active and Latent Tuberculosis and Are Associated with Treatment Responses

Background

Biomarkers to differentiate between active tuberculosis (TB) and latent TB infection (LTBI) and to monitor treatment responses are requested to complement TB diagnostics and control, particularly in patients with multi-drug resistant TB. We have studied soluble markers of the Toll-like-receptor 4 (TLR-4) pathway in various stages of TB disease and during anti-TB treatment.

Methods

Plasma samples from patients with culture confirmed drug-sensitive TB (n = 19) were collected before and after 2, 8 and 24 weeks of efficient anti-TB treatment and in a LTBI group (n = 6). Soluble (s) CD14 and myeloid differentiation-2 (MD-2) were analyzed by the Enzyme-linked immunosorbent assay (ELISA). Lipopolysaccharide (LPS) was analyzed by the Limulus Amebocyte Lysate colorimetric assay. Nonparametric statistics were applied.

Results

Plasma levels of sCD14 (p<0.001), MD-2 (p = 0.036) and LPS (p = 0.069) were elevated at baseline in patients with untreated active TB compared to the LTBI group. MD-2 concentrations decreased after 2 weeks of treatment (p = 0.011), while LPS levels decreased after 8 weeks (p = 0.005). In contrast, sCD14 levels increased after 2 weeks (p = 0.047) with a subsequent modest decrease throughout the treatment period. There was no significant difference in concentrations of any of these markers between patients with pulmonary and extrapulmonary TB or between patients with or without symptoms.

Conclusion

Our data suggest that plasma levels of LPS, MD-2 and sCD14 can discriminate between active TB and LTBI. A decline in LPS and MD-2 concentrations was associated with response to anti-TB treatment. The clinical potential of these soluble TLR-4 pathway proteins needs to be further explored.