Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K⁺ selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of ⁸⁶Rb⁺ into chromaffin granules prepared from bovine adrenal gland medulla. The ⁸⁶Rb⁺ influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432±9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca²⁺-activated K⁺ channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

A modified dilution method reveals higher protozoan growth rates than the size fractionation method

Protozoan growth rates are typically estimated with the size fractionation method in which the organisms studied are separated from the larger grazers. This method was compared with a specially modified dilution method which removed not only the grazing pressure of larger grazers but also that of predators of the same size as the organisms studied. Five comparisons were performed under natural conditions with the following marine protozoa: Balanion comatum (3 sets of data), Ebria tripartita, and Strombidium sp. The dilution method revealed significantly higher growth rates (Wilcoxon's signed-rank test, p = 0.04), which underscores the importance of predation between organisms of the same size. It directly demonstrated that the size fractionation method may significantly underestimate protozoan growth rates.     

Chemerin Is an Antimicrobial Agent in Human Epidermis

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val⁶⁶-Pro⁸⁵, which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.     

Dysbiosis of upper respiratory tract microbiota in elderly pneumonia patients

Bacterial pneumonia is a major cause of morbidity and mortality in elderly. We hypothesize that dysbiosis between regular residents of the upper respiratory tract (URT) microbiome, that is balance between commensals and potential pathogens, is involved in pathogen overgrowth and consequently disease. We compared oropharyngeal microbiota of elderly pneumonia patients (n=100) with healthy elderly (n=91) by 16S-rRNA-based sequencing and verified our findings in young adult pneumonia patients (n=27) and young healthy adults (n=187). Microbiota profiles differed significantly between elderly pneumonia patients and healthy elderly (PERMANOVA, P<0.0005). Highly similar differences were observed between microbiota profiles of young adult pneumonia patients and their healthy controls. Clustering resulted in 11 (sub)clusters including 95% (386/405) of samples. We observed three microbiota profiles strongly associated with pneumonia (P<0.05) and either dominated by lactobacilli (n=11), Rothia (n=51) or Streptococcus (pseudo)pneumoniae (n=42). In contrast, three other microbiota clusters (in total n=183) were correlated with health (P<0.05) and were all characterized by more diverse profiles containing higher abundances of especially Prevotella melaninogenica, Veillonella and Leptotrichia. For the remaining clusters (n=99), the association with health or disease was less clear. A decision tree model based on the relative abundance of five bacterial community members in URT microbiota showed high specificity of 95% and sensitivity of 84% (89% and 73%, respectively, after cross-validation) for differentiating pneumonia patients from healthy individuals. These results suggest that pneumonia in elderly and young adults is associated with dysbiosis of the URT microbiome with bacterial overgrowth of single species and absence of distinct anaerobic bacteria. Whether the observed microbiome changes are a cause or a consequence of the development of pneumonia or merely coincide with disease status remains a question for future research.     

Pregnancy and Delivery in Patients with Mastocytosis Treated at the Polish Center of the European Competence Network on Mastocytosis (ECNM)

Objective

To present current guidelines regarding treatment of mastocytosis in pregnancy on the example of observed patients.

Design

Case control national study.

Setting

Polish Center of the European Competence Network on Mastocytosis (ECNM).

Population or Sample

23 singleton spontaneous pregnancies in 17 women diagnosed with mastocytosis in years 1999–2014, before becoming pregnant.

Methods

Prospective analysis outcomes of pregnancies and deliveries.

Main Outcome Measures

Survey developed in cooperation with the Spanish Instituto de Estudios de Mastocitosis de Castilla-La Mancha (CLMast), Hospital Virgen del Valle, Toledo, Red Espańola de Mastocitosis (REMA), Spain.

Results

All 23 pregnancies resulted from natural conception. Obstetrical complications recorded in the first trimester included spontaneous miscarriage (5 pregnancies). Four patients delivered preterm, including one delivery due to preeclampsia at 26 weeks which resulted with neonate death due to extreme prematurity. Five women delivered via cesarean: four due to obstetrical indications and one due to mastocytosis, during which no anesthesia related complications were recorded. Of patients delivering vaginally, two received extradural anesthesia, three required oxytocin infusion due to uterine hypotonia. No labor complications were recorded. In one woman with pregnancy-induced hypertension, early puerperium was complicated by the presence of persistent arterial hypertension. One neonate was born with the signs of cutaneous mastocytosis. Another neonate was diagnosed with Patau syndrome. Four women were treated for mastocytosis prior to conception and continued therapy after becoming pregnant. One patient was put on medications in the first trimester due to worsening of her symptoms. Pregnancy exerted only a slight effect on the intensity and frequency of mastocytosis-related symptoms observed. Worsening of the disease-related symptoms was documented in only four patients (23%). None of the patients showed the signs of anaphylaxis, either before becoming pregnant, or during pregnancy and puerperium.

Conclusions

There is no contraindication to pregnancy when mastocystosis-related pathologies are under appropriate medical control.