The surfactant protein C mutation A116D alters cellular processing, stress tolerance, surfactant lipid composition, and immune cell activation

<p>Abstract</p> <p>Background</p> <p>Surfactant protein C (SP-C) is important for the function of pulmonary surfactant. Heterozygous mutations in <it>SFTPC</it>, the gene encoding SP-C, cause sporadic and familial interstitial lung disease (ILD) in children and adults. Mutations mapping to the BRICHOS domain located within the SP-C proprotein result in perinuclear aggregation of the proprotein. In this study, we investigated the effects of the mutation A116D in the BRICHOS domain of SP-C on cellular homeostasis. We also evaluated the ability of drugs currently used in ILD therapy to counteract these effects.</p> <p>Methods</p> <p>SP-C^A116Dwas expressed in MLE-12 alveolar epithelial cells. We assessed in vitro the consequences for cellular homeostasis, immune response and effects of azathioprine, hydroxychloroquine, methylprednisolone and cyclophosphamide.</p> <p>Results</p> <p>Stable expression of SP-C^A116Din MLE-12 alveolar epithelial cells resulted in increased intracellular accumulation of proSP-C processing intermediates. SP-C^A116Dexpression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-C^A116Dcells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4⁺lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-C^A116Don neighboring cells in the alveolar space.</p> <p>Conclusions</p> <p>We show that the A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light on the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act.</p

Neutrophil Elastase Enhances Sputum Solubilization in Cystic Fibrosis Patients Receiving DNase Therapy

Cystic fibrosis patients suffer from chronic lung infection and inflammation due to the secretion of viscous sputum. Sputum viscosity is caused by extracellular DNA, some of which originates from the release of neutrophil extracellular traps (NETs). During NET formation neutrophil elastase (NE) partially processes histones to decondense chromatin. NE is abundant in CF sputum and is thought to contribute to tissue damage. Exogenous nucleases are a palliative treatment in CF as they promote sputum solubilization. We show that in a process reminiscent of NET formation, NE enhances sputum solubilization by cleaving histones to enhance the access of exogenous nucleases to DNA. In addition, we find that in Cf sputum NE is predominantly bound to DNA, which is known to downregulate its proteolytic activity and may restrict host tissue damage. The beneficial role of NE in CF sputum solubilization may have important implications for the development of CF therapies targeting NE

Diverging Mechanisms of Activation of Chemokine Receptors Revealed by Novel Chemokine Agonists

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions

Common HLA Alleles Associated with Health, but Not with Facial Attractiveness

Three adaptive hypotheses have been proposed to explain the link between the human leucocyte antigen (HLA) genes, health measures and facial attractiveness: inbreeding avoidance, heterozygote advantage and frequency-dependent selection. This paper reports findings that support a new hypothesis relating HLA to health. We suggest a new method to quantify the level of heterozygosity. HLA heterozygosity did not significantly predict health measures in women, but allele frequency did. Women with more common HLA alleles reported fewer cold and flu bouts per year, fewer illnesses in the previous year and rated themselves healthier than women with rare alleles. To our knowledge, this is the first study to show a positive correlation between HLA allele frequency and general health measures. We propose that certain common HLA alleles confer resistance to prevalent pathogens. Nevertheless, neither HLA heterozygosity nor allele frequency significantly predicted how healthy or attractive men rated the female volunteers. Three non-mutually exclusive explanations are put forward to explain this finding

Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

<p>Abstract</p> <p>Background</p> <p>Strains of <it>Plasmodium falciparum </it>genetically resistant to chloroquine (CQ) due to the presence of <it>pfcrt </it>76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant <it>P. falciparum </it>isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect <it>pfcrt </it>76T-bearing <it>P. falciparum </it>minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.</p> <p>Methods</p> <p>This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect <it>pfcrt </it>76T-bearing minority variant <it>P. falciparum</it>. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.</p> <p>Results</p> <p>Thirty one clinical <it>P. falciparum </it>isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the <it>pfcrt </it>K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant <it>P. falciparum </it>strains after therapy as detected by HTA. RFLP analysis failed to detect any <it>pfcrt </it>K76T-bearing isolates.</p> <p>Conclusion</p> <p>These findings indicate that genetically CQ-resistant <it>P. falciparum </it>are more common than previously thought in Madagascar even though the fitness of the minority variant <it>pfcrt </it>76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.</p

Proportionality between variances in gene expression induced by noise and mutation: consequence of evolutionary robustness

<p>Abstract</p> <p>Background</p> <p>Characterization of robustness and plasticity of phenotypes is a basic issue in evolutionary and developmental biology. The robustness and plasticity are concerned with changeability of a biological system against external perturbations. The perturbations are either genetic, i.e., due to mutations in genes in the population, or epigenetic, i.e., due to noise during development or environmental variations. Thus, the variances of phenotypes due to genetic and epigenetic perturbations provide quantitative measures for such changeability during evolution and development, respectively.</p> <p>Results</p> <p>Using numerical models simulating the evolutionary changes in the gene regulation network required to achieve a particular expression pattern, we first confirmed that gene expression dynamics robust to mutation evolved in the presence of a sufficient level of transcriptional noise. Under such conditions, the two types of variances in the gene expression levels, i.e. those due to mutations to the gene regulation network and those due to noise in gene expression dynamics were found to be proportional over a number of genes. The fraction of such genes with a common proportionality coefficient increased with an increase in the robustness of the evolved network. This proportionality was generally confirmed, also under the presence of environmental fluctuations and sexual recombination in diploids, and was explained from an evolutionary robustness hypothesis, in which an evolved robust system suppresses the so-called error catastrophe - the destabilization of the single-peaked distribution in gene expression levels. Experimental evidences for the proportionality of the variances over genes are also discussed.</p> <p>Conclusions</p> <p>The proportionality between the genetic and epigenetic variances of phenotypes implies the correlation between the robustness (or plasticity) against genetic changes and against noise in development, and also suggests that phenotypic traits that are more variable epigenetically have a higher evolutionary potential.</p

Resurrection of an ancestral 5S rRNA

<p>Abstract</p> <p>Background</p> <p>In addition to providing phylogenetic relationships, tree making procedures such as parsimony and maximum likelihood can make specific predictions of actual historical sequences. Resurrection of such sequences can be used to understand early events in evolution. In the case of RNA, the nature of parsimony is such that when applied to multiple RNA sequences it typically predicts ancestral sequences that satisfy the base pairing constraints associated with secondary structure. The case for such sequences being actual ancestors is greatly improved, if they can be shown to be biologically functional.</p> <p>Results</p> <p>A unique common ancestral sequence of 28 <it>Vibrio </it>5S ribosomal RNA sequences predicted by parsimony was resurrected and found to be functional in the context of the <it>E. coli </it>cellular environment. The functionality of various point variants and intermediates that were constructed as part of the resurrection were examined in detail. When separately introduced the changes at single stranded positions and individual double variants at base-paired positions were also viable. An additional double variant was examined at a different base-paired position and it was also valid.</p> <p>Conclusions</p> <p>The results show that at least in the case of the 5S rRNAs considered here, ancestors predicted by parsimony are likely to be realistic when the prediction is not overly influenced by single outliers. It is especially noteworthy that the phenotype of the predicted ancestors could be anticipated as a cumulative consequence of the phenotypes of the individual variants that comprised them. Thus, point mutation data is potentially useful in evaluating the reasonableness of ancestral sequences predicted by parsimony or other methods. The results also suggest that in the absence of significant tertiary structure constraints double variants that preserve pairing in stem regions will typically be accepted. Overall, the results suggest that it will be feasible to resurrect additional meaningful 5S rRNA ancestors as well as ancestral sequences of many different types of RNA.</p

Incompetence of Neutrophils to Invasive Group A streptococcus Is Attributed to Induction of Plural Virulence Factors by Dysfunction of a Regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection