Impaired innate interferon induction in severe therapy resistant atopic asthmatic children

Deficient type I interferon-β and type III interferon-λ induction by rhinoviruses has previously been reported in mild/moderate atopic asthmatic adults. No studies have yet investigated if this occurs in severe therapy resistant asthma (STRA). Here, we show that compared with non-allergic healthy control children, bronchial epithelial cells cultured ex vivo from severe therapy resistant atopic asthmatic children have profoundly impaired interferon-β and interferon-λ mRNA and protein in response to rhinovirus (RV) and polyIC stimulation. Severe treatment resistant asthmatics also exhibited increased virus load, which negatively correlated with interferon mRNA levels. Furthermore, uninfected cells from severe therapy resistant asthmatic children showed lower levels of Toll-like receptor-3 mRNA and reduced retinoic acid inducible gene and melanoma differentiation-associated gene 5 mRNA after RV stimulation. These data expand on the original work, suggesting that the innate anti-viral response to RVs is impaired in asthmatic tissues and demonstrate that this is a feature of STRA.

Romany Families in Romania: Intelligence and Personality of Children Cross-cultural and Specific Problems of Development

Psychogenetic research has emphasised the influence of social factors on a child's intellectual development. In her work, Ms. Dumitrascu examines two such factors; family size and order of birth. However, since these formal parameters tend to be unstable, other more informal factors should be taken into consideration. Of these, perhaps the most interesting is the "style" of parental education, which Ms. Dumitrascu regards as an expression of national traditions at the family level. This educational style is culture dependent. Only a comparative, cross-cultural study can reveal the real mechanism through which educational style influences the development of a child's intellect and personality. Ms. Dumitrascu conducted an experimental cross-cultural study aimed at examining the effects of the family environment on a child's intellectual development. Three distinct populations were involved in her investigation, each having quite a distinct status in their geographical area; Romanians, Romanies (Gypsies) from Romania, and Russians from the Republic of Moldova. She presented her research in the form of a series of articles written in English totalling 85 pages, and also on disc. A significant difference was revealed between the intelligence of a child living in a large family, and that of a child with no brothers or sisters. In the case of Romany children, the gap is remarkably large. Ms. Dumitrascu concludes that the simultaneous action of several negative factors (low socio-economic status, large family size, socio-cultural isolation of a population) may delay child development. Subjected to such a precarious environment, Romany children do not seek self-realisation, but rather struggle to survive the hardship. Most of them remain out of civilisation. Unfortunately, adult Romanies seldom express any concern regarding their children's successful social integration. The school as main socialisation tool has no value for most parents. Ms. Dumitrascu argues the need for a major effort aimed at helping Romany's social integration. She hopes this project will be of some help for psychologists, social workers, teachers, and all those who are interested in the integration into society of minority groups.

Sonographic Assessment of the Umbilical Cord

The umbilical cord is not an inert structure, suspended between the fetus and placenta, but it plays an active role and it is involved in several processes afflicting the feto-placental unit. Its study has to be regarding not only its morphology and morphometry, and the impendance of blood flow by Doppler waveform analysis, but it includes also an analysis of the coiling type and the amount of the Wharton Jelly. The umbilical cord has been considered like an important and huge source of informations, useful to assess the well-being of the fetus and the outcome of pregnancy. The standardization of ultrasound techniques is the first step to speak the same language and make the study of this structure a fundamental part of well-being fetus assessment. This article is carefully focused on morphologic, morphometric and functional ultrasound examination of umbilical cord and suggests that any anomaly detected should provide an indication for an intense fetal follow-up, useful for early helpful therapy, preventing serious complication for the pregnancy.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Targets of emerging therapies for viral hepatitis B and C

Viral hepatitis B and C, structurally two completely different viruses, commonly infect human hepatocytes and cause similar clinical manifestations. Since their discovery, IFN has been a pillar in the treatment. However, because of the different natures of the viruses, therapeutic approaches diverge and new treatment targets are tailored specifically for each virus. Herein, the authors analyse therapeutic approaches for hepatitis B virus (HBV) and hepatitis C virus (HCV) and focus on emerging concepts that are under clinical evaluation. In particular, promising viral inhibitors for HBV and HCV are reviewed and the current status of research for gene therapy for HCV is described. Immune therapy is a fast-moving field with fascinating results which include therapeutic vaccines and toll-like receptor agonists that could improve tomorrow's treatment approaches.

Late-onset manifestation of antenatal Bartter syndrome as a result of residual function of the mutated renal Na+-K+-2Cl- co-transporter

Genetic defects of the Na+-K+-2Cl- (NKCC2) sodium potassium chloride co-transporter result in severe, prenatal-onset renal salt wasting accompanied by polyhydramnios, prematurity, and life-threatening hypovolemia of the neonate (antenatal Bartter syndrome or hyperprostaglandin E syndrome). Herein are described two brothers who presented with hyperuricemia, mild metabolic alkalosis, low serum potassium levels, and bilateral medullary nephrocalcinosis at the ages of 13 and 15 yr. Impaired function of sodium chloride reabsorption along the thick ascending limb of Henle's loop was deduced from a reduced increase in diuresis and urinary chloride excretion upon application of furosemide. Molecular genetic analysis revealed that the brothers were compound heterozygotes for mutations in the SLC12A1 gene coding for the NKCC2 co-transporter. Functional analysis of the mutated rat NKCC2 protein by tracer-flux assays after heterologous expression in Xenopus oocytes revealed significant residual transport activity of the NKCC2 p.F177Y mutant construct in contrast to no activity of the NKCC2-D918fs frameshift mutant construct. However, coexpression of the two mutants was not significantly different from that of NKCC2-F177Y alone or wild type. Membrane expression of NKCC2-F177Y as determined by luminometric surface quantification was not significantly different from wild-type protein, pointing to an intrinsic partial transport defect caused by the p.F177Y mutation. The partial function of NKCC2-F177Y, which is not negatively affected by NKCC2-D918fs, therefore explains a mild and late-onset phenotype and for the first time establishes a mild phenotype-associated SLC12A1 gene mutation.

Becker muscular dystrophy with marked divergence between clinical and molecular genetic findings: case series

Both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations of the X-linked dystrophin gene. BMD patients are less affected clinically than DMD patients. We present five patients with a diagnosis of BMD. First, two identical twins, with a deletion of exon 48 of the dystrophin gene, who experienced prominent muscle cramps from the age of three. The histopathological examination of muscle biopsies of these two twins revealed only very slight muscle fiber alterations. Second, two brothers who displayed marked, unusual intrafamilial variability of the clinical picture as well as showing a new point mutation in the dystrophin gene. And finally, a fifth boy who displayed a new point mutation in the dystrophin gene. Although he was clinically asymptomatic at the age of 15 and muscle biopsy only showed very minor myopathic signs, serum Creatine Kinase (CK) levels had been considerably elevated for years. Taken together, these cases add to the spectrum of marked discrepancies in clinical, histopathological and molecular genetic findings in BMD.

Mitochondrial neurogastrointestinal encephalomyopathy in three siblings: clinical, genetic and neuroradiological features

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder in which a nuclear mutation of the thymidine phosphorylase (TP) gene causes mitochondrial genomic dysfunction. Patients suffer from gastrointestinal dysmotility, cachexia, ptosis, external ophthalmoparesis, myopathy and polyneuropathy. Magnetic resonance imaging (MRI) shows leukoencephalopathy. We describe clinical, genetic and neuroradiological features of three brothers affected with MNGIE. Clinical examination, laboratory analyses, MRI and magnetic resonance spectroscopy (MRS) of the brain, and genetic analysis have been performed in all six members of the family with the three patients with MNGIE. Two of them are monozygous twins. They all suffered from gastrointestinal dysmotility, cachexia, ophthalmoplegia, muscular atrophies, and polyneuropathy. Urinary thymidine was elevated in the patients related to the severity of clinical disease, and urinary thymidine (normally not detectable) was also found in a heterozygous carrier. Brain MRI showed leukoencephalopathy in all patients; however, their cognitive functioning was normal. Brain MRS demonstrated reduced N-acetylaspartate and choline in severely affected areas. MRI of heterozygous carriers was normal. A new mutation (T92N) in the TP gene was identified. Urinary thymidine is for the first time reported to be detectable in a heterozygous carrier. MRS findings indicate loss of neurons, axons, and glial cells in patients with MNGIE, but not in heterozygous carriers.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Pseudodominant Inheritance of Goitrous Congenital Hypothyroidism Caused by TPO Mutations: Molecular and in Silico Studies

Context and Objective: Most cases of goitrous congenital hypothyroidism (CH) from thyroid dyshormonogenesis 1) follow a recessive mode of inheritance and 2) are due to mutations in the thyroid peroxidase gene (TPO). We report the genetic mechanism underlying the apparently dominant inheritance of goitrous CH in a nonconsanguineous family of French Canadian origin. Design, Setting, and Participants: Two brothers identified by newborn TSH screening had severe hypothyroidism and a goiter with increased (99m)Tc uptake. The mother was euthyroid, but the father and two paternal uncles had also been diagnosed with goitrous CH. After having excluded PAX8 gene mutations, we hypothesized that the underlying defect could be TPO mutations. Results: Both compound heterozygous siblings had inherited a mutant TPO allele carried by their mother (c.1496delC; p.Pro499Argfs2X), and from their father, one brother had inherited a missense mutation (c.1978C-->G; p.Gln660Glu) and the other an insertion (c.1955insT; p.Phe653Valfs15X). The thyroid gland of one uncle who is a compound heterozygote for TPO mutations (p.Phe653Valfs15X/p.Gln660Glu) was removed because of concurrent multiple endocrine neoplasia type 2A. Immunohistochemistry revealed normal TPO staining, implying that Gln660Glu TPO is expressed properly. Modeling of this mutant in silico suggests that its three-dimensional structure is conserved, whereas the electrostatic binding energy between the Gln660Glu TPO and its heme group becomes repulsive. Conclusion: We report a pedigree presenting with pseudodominant goitrous CH due to segregation of three different TPO mutations. Although goitrous CH generally follows a recessive mode of inheritance, the high frequency of TPO mutations carriers may lead to pseudodominant inheritance.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Association of lipopolysaccharide-binding protein and coronary artery disease in men

OBJECTIVES: In this study we tested the hypothesis that lipopolysaccharide-binding protein (LBP) might be able to be used as a biomarker for coronary artery disease (CAD). BACKGROUND: The mechanisms by which the innate immune recognition of pathogens could lead to atherosclerosis remain unclear. Lipopolysaccharide-binding protein is the first protein to encounter lipopolysaccharide and to deliver it to its cellular targets, toll-like receptors; therefore, its presence might be a reliable biomarker that indicates activation of innate immune responses. METHODS: A total of 247 men undergoing elective coronary angiography were studied, and the extent of coronary atherosclerosis was assessed by 2 established scores: "extent score" and "severity score." Levels of LBP, markers of inflammation, and traditional risk factors for CAD were assessed. RESULTS: Serum LBP concentration was significantly increased in 172 patients with angiographically confirmed CAD compared with 75 individuals without coronary atherosclerosis (20.6 +/- 8.7 pg/ml vs. 17.1 +/- 6.0 pg/ml, respectively; p = 0.002). Moreover in multivariable logistic regression analyses, adjusted for established cardiovascular risk factors and markers of systemic inflammation, LBP was a significant and independent predictor of prevalent CAD (p < 0.05 in all models). CONCLUSIONS: Lipopolysaccharide-binding protein might serve as a novel marker for CAD in men. The present results underlie the potential importance of innate immune mechanisms for CAD. Further studies are warranted to bolster the data and to identify pathogenetic links between innate immune system activation and atherosclerosis.

Adjuvant TACE inhibitor treatment improves the outcome of TLR2-/- mice with experimental pneumococcal meningitis

BACKGROUND: Streptococcus (S.) pneumoniae meningitis has a high lethality despite antibiotic treatment. Inflammation is a major pathogenetic factor, which is unresponsive to antibiotics. Therefore adjunctive therapies with antiinflammatory compounds have been developed. TNF484 is a TNF-alpha converting enzyme (TACE) inhibitor and has been found efficacious in experimental meningitis. Toll-like receptor 2 (TLR2) contributes to host response in pneumococcal meningitis by enhancing bacterial clearing and downmodulating inflammation. In this study, TNF484 was applied in mice, which lacked TLR2 and exhibited a strong meningeal inflammation. METHODS: 103 CFU S. pneumoniae serotype 3 was inoculated subarachnoidally into C57BL/6 wild type (wt) mice or TLR2-/-, CD14-/- and CD14-/-/TLR2-/- mice. Severity of disease and survival was followed over 9 days. Response to antibiotics (80 mg/kg ceftriaxone i.p. for 5 days) and/or TACE inhibitor treatment (1 mg/kg s.c. twice daily for 4 days) was evaluated. Animals were sacrificed after 12, 24, and 48 h for analysis of bacterial load in cerebrospinal fluid (CSF) and brain and for TNF and leukocyte measurements in CSF. RESULTS: TLR2-/- mice were significantly sicker than the other mouse strains 24 h after infection. All knockout mice showed higher disease severity after 48 h and died earlier than wt mice. TNF release into CSF was significantly more elevated in TLR2-/- than in the other strains after 24 h. Brain bacterial numbers were significantly higher in all knockout than wt mice after 24 h. Modulation of outcome by antibiotic and TACE inhibitor treatment was evaluated. With antibiotic therapy all wt, CD14-/- and TLR2-/-/CD14-/- mice, but only 79% of TLR2-/- mice, were rescued. TACE inhibitor treatment alone did not rescue, but prolonged survival in wt mice, and in TLR2-/- and CD14-/- mice to the values observed in untreated wt mice. By combined antibiotic and TACE inhibitor treatment 95% of TLR2-/- mice were rescued. CONCLUSION: During pneumococcal meningitis strong inflammation in TLR2-deficiency was associated with incomplete responsiveness to antibiotics and complete response to combined antibiotic and TACE inhibitor treatment. TACE inhibitor treatment offers a promising adjuvant therapeutic strategy in pneumococcal meningitis.

Using distinct molecular signatures of human monocytes and dendritic cells to predict adjuvant activity and pyrogenicity of TLR agonists

We present a systematic study that defines molecular profiles of adjuvanticity and pyrogenicity induced by agonists of human Toll-like receptor molecules in vitro. Using P(3)CSK(4), Lipid A and Poly I:C as model adjuvants we show that all three molecules enhance the expansion of IFNgamma(+)/CD4(+) T cells from their naïve precursors following priming with allogeneic DC in vitro. In contrast, co-culture of naive CD4(+) T cells with allogeneic monocytes and TLR2/TLR4 agonists only resulted in enhanced T cell proliferation. Distinct APC molecular signatures in response to each TLR agonist underline the dual effect observed on T cell responses. Using protein and gene expression assays, we show that TNF-alpha and CXCL10 represent DC-restricted molecular signatures of TLR2/TLR4 and TLR3 activation, respectively, in sharp contrast to IL-6 produced by monocytes upon stimulation with P(3)CSK(4) and Lipid A. Furthermore, although all TLR agonists are able to up-regulate proIL-1beta specific gene in both cell types, only monocyte activation with Lipid A results in detectable IL-1beta release. These molecular profiles, provide a simple screen to select new immune enhancers of human Th1 responses suitable for clinical application.