Protective effects of lipocalin-2 (LCN2) in acute liver injury suggest a novel function in liver homeostasis

Lipocalin-2 is expressed under pernicious conditions such as intoxication, infection, inflammation and other forms of cellular stress. Experimental liver injury induces rapid and sustained LCN2 production by injured hepatocytes. However, the precise biological function of LCN2 in liver is still unknown. In this study, LCN2^?/? mice were exposed to short term application of CCl₄, lipopolysaccharide and Concanavalin A, or subjected to bile duct ligation. Subsequent injuries were assessed by liver function analysis, qRT-PCR for chemokine and cytokine expression, liver tissue Western blot, histology and TUNEL assay. Serum LCN2 levels from patients suffering from liver disease were assessed and evaluated. Acute CCl₄ intoxication showed increased liver damage in LCN2^?/? mice indicated by higher levels of aminotransferases, and increased expression of inflammatory cytokines and chemokines such as IL-1β, IL-6, TNF-α and MCP-1/CCL2, resulting in sustained activation of STAT1, STAT3 and JNK pathways. Hepatocytes of LCN2^?/? mice showed lipid droplet accumulation and increased apoptosis. Hepatocyte apoptosis was confirmed in the Concanavalin A and lipopolysaccharide models. In chronic models (4 weeks bile duct ligation or 8 weeks CCl₄ application), LCN2^?/? mice showed slightly increased fibrosis compared to controls. Interestingly, serum LCN2 levels in diseased human livers were significantly higher compared to controls, but no differences were observed between cirrhotic and non-cirrhotic patients. Upregulation of LCN2 is a reliable indicator of liver damage and has significant hepato-protective effect in acute liver injury. LCN2 levels provide no correlation to the degree of liver fibrosis but show significant positive correlation to inflammation instead.     

Coccidioidomycosis and blastomycosis: Advances in molecular diagnosis

Clinical isolates of Coccidioides spp. and Blastomyces dermatitidis can be identified by chemiluminescent DNA probes and PCR assays targeting multicopy genes. In fixed tissue samples, cells of the two fungi are specified by in situ hybridization and PCR assays targeting 18S rDNA but sequencing of the products is mandatory. Nested PCR assays targeting genes encoding species- or genus-specific proteins like proline rich antigen of Coccidioides spp. and B. dermatitidis adhesin facilitate amplification of specific DNA from fixed tissue samples. The value of DNA amplification from native specimens of suspected cases of coccidioidomycosis or blastomycosis still needs to be determined.     

Conserved roles of ems/Emx and otd/Otx genes in olfactory and visual system development in Drosophila and mouse

The regional specialization of brain function has been well documented in the mouse and fruitfly. The expression of regulatory factors in specific regions of the brain during development suggests that they function to establish or maintain this specialization. Here, we focus on two such factors—the Drosophila cephalic gap genes empty spiracles (ems) and orthodenticle (otd), and their vertebrate homologues Emx1/2 and Otx1/2—and review novel insight into their multiple crucial roles in the formation of complex sensory systems. While the early requirement of these genes in specification of the neuroectoderm has been discussed previously, here we consider more recent studies that elucidate the later functions of these genes in sensory system formation in vertebrates and invertebrates. These new studies show that the ems and Emx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective olfactory systems. Moreover, they demonstrate that the otd and Otx genes in both flies and mice are essential for the development of the peripheral and central neurons of their respective visual systems. Based on these recent experimental findings, we discuss the possibility that the olfactory and visual systems of flies and mice share a common evolutionary origin, in that the conserved visual and olfactory circuit elements derive from conserved domains of otd/Otx and ems/Emx action in the urbilaterian ancestor.     

Association of a common TLR-6 polymorphism with coronary artery disease – implications for healthy ageing?

Background

The pro-inflammatory status of the elderly triggers most of the age-related diseases such as cancer and atherosclerosis. Atherosclerosis, the leading cause world wide of morbidity and death, is an inflammatory disease influenced by life-style and genetic host factors. Stimuli such as oxLDL or microbial ligands have been proposed to trigger inflammation leading to atherosclerosis. It has recently been shown that oxLDL activates immune cells via the Toll-like receptor (TLR) 4/6 complex. Several common single nucleotide polymorphisms (SNPs) of the TLR system have been associated with atherosclerosis. To investigate the role of TLR-6 we analyzed the association of the TLR-6 SNP Pro249Ser with atherogenesis.

Results

Genotyping of two independent groups with CAD, as well as of healthy controls revealed a significant association of the homozygous genotype with a reduced risk for atherosclerosis (odds ratio: 0.69, 95% CI 0.51-0.95, P?=?0.02). In addition, we found a trend towards an association with the risk of restenosis after transluminal coronary angioplasty (odds ratio: 0.53, 95% CI 0.24-1.16, P?=?0.12). In addition, first evidence is presented that the frequency of this protective genotype increases in a healthy population with age. Taken together, our results define a role for TLR-6 and its genetic variations in modulating the inflammatory response leading to atherosclerosis.

Conclusions

These results may lead to a better risk stratification, and potentially to an improved prophylactic treatment of high-risk populations. Furthermore, the protective effect of this polymorphism may lead to an increase of this genotype in the healthy elderly and may therefore be a novel genetic marker for the well-being during aging.

     

The Effect of New Zealand Kanuka,Manuka and Clover Honeys on Bacterial Growth Dynamics and Cellular Morphology Varies According to the Species

Treatment of chronic wounds is becoming increasingly difficult due to antibiotic resistance. Complex natural products with antimicrobial activity, such as honey, are now under the spotlight as alternative treatments to antibiotics. Several studies have shown honey to have broad-spectrum antibacterial activity at concentrations present in honey dressings, and resistance to honey has not been attainable in the laboratory. However not all honeys are the same and few studies have used honey that is well defined both in geographic and chemical terms. Here we have used a range of concentrations of clover honey and a suite of manuka and kanuka honeys from known geographical locations, and for which the floral source and concentration of methylglyoxal and hydrogen peroxide potential were defined, to determine their effect on growth and cellular morphology of four bacteria: Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. While the general trend in effectiveness of growth inhibition was manuka>manuka-kanuka blend>kanuka>clover, the honeys had varying and diverse effects on the growth and cellular morphology of each bacterium, and each organism had a unique response profile to these honeys. P. aeruginosa showed a markedly different pattern of growth inhibition to the other three organisms when treated with sub-inhibitory concentrations of honey, being equally sensitive to all honeys, including clover, and the least sensitive to honey overall. While hydrogen peroxide potential contributed to the antibacterial activity of the manuka and kanuka honeys, it was never essential for complete growth inhibition. Cell morphology analysis also showed a varied and diverse set of responses to the honeys that included cell length changes, cell lysis, and alterations to DNA appearance. These changes are likely to reflect the different regulatory circuits of the organisms that are activated by the stress of honey treatment.     

Generation of Amyloid-β Is Reduced by the Interaction of Calreticulin with Amyloid Precursor Protein,Presenilin and Nicastrin

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer''s disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca²⁺- and N-glycan-independent interaction is mediated by amino acids 330–344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β₄₂ levels in culture supernatants, while transfection with the P-domain increases amyloid-β₄₀ levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330–344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β₄₀ and amyloid-β₄₂ levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer''s disease.     

Grouping by Proximity in Haptic Contour Detection

We investigated the applicability of the Gestalt principle of perceptual grouping by proximity in the haptic modality. To do so, we investigated the influence of element proximity on haptic contour detection. In the course of four sessions ten participants performed a haptic contour detection task in which they freely explored a haptic random dot display that contained a contour in 50% of the trials. A contour was defined by a higher density of elements (raised dots), relative to the background surface. Proximity of the contour elements as well as the average proximity of background elements was systematically varied. We hypothesized that if proximity of contour elements influences haptic contour detection, detection will be more likely when contour elements are in closer proximity. This should be irrespective of the ratio with the proximity of the background elements. Results showed indeed that the closer the contour elements were, the higher the detection rates. Moreover, this was the case independent of the contour/background ratio. We conclude that the Gestalt law of proximity applies to haptic contour detection.     

Hyaluronic Acid Suppresses the Expression of Metalloproteinases in Osteoarthritic Cartilage Stimulated Simultaneously by Interleukin 1β and Mechanical Load

Purpose

In patients with osteoarthritis (OA), intraarticular injection of hyaluronic acid (HA) frequently results in reduced pain and improved function for prolonged periods of time, i.e. more than 6 months. However, the mechanisms underlying these effects are not fully understood. Our underlying hypothesis is that HA modifies the enzymatic breakdown of joint tissues.

Methods

To test this hypothesis, we examined osteochondral cylinders from 12 OA patients. In a bioreactor, these samples were stimulated by interleukin 1β (Il1ß) (2 ng/ml) plus mechanical load (2.0 Mpa at 0.5 Hz horizontal and 0.1 Hz vertical rotation), thus the experimental setup recapitulated both catabolic and anabolic clues of the OA joint.

Results

Upon addition of HA at either 1 or 3 mg/ml, we observed a significant suppression of expression of metalloproteinase (MMP)-13. A more detailed analysis based on the Kellgren and Lawrence (K&L) OA grade, showed a much greater degree of suppression of MMP-13 expression in grade IV as compared to grade II OA. In contrast to the observed MMP-13 suppression, treatment with HA resulted in a suppression of MMP-1 expression only at 1 mg/ml HA, while MMP-2 expression was not significantly affected by either HA concentration.

Conclusion

Together, these data suggest that under concurrent catabolic and anabolic stimulation, HA exhibits a pronounced suppressive effect on MMP-13. In the long-run these findings may benefit the development of treatment strategies aimed at blocking tissue degradation in OA patients.     

Microparticle-Induced Coagulation Relates to Coronary Artery Atherosclerosis in Severe Aortic Valve Stenosis

Background

Circulating microparticles (MPs) derived from endothelial cells and blood cells bear procoagulant activity and promote thrombin generation. Thrombin exerts proinflammatory effects mediating the progression of atherosclerosis. Aortic valve stenosis may represent an atherosclerosis-like process involving both the aortic valve and the vascular system. The aim of this study was to investigate whether MP-induced thrombin generation is related to coronary atherosclerosis and aortic valve calcification.

Methods

In a cross-sectional study of 55 patients with severe aortic valve stenosis, we assessed the coronary calcification score (CAC) as indicator of total coronary atherosclerosis burden, and aortic valve calcification (AVC) by computed tomography. Thrombin-antithrombin complex (TATc) levels were measured as a marker for thrombin formation. Circulating MPs were characterized by flow cytometry according to the expression of established surface antigens and by measuring MP-induced thrombin generation.

Results

Patients with CAC score below the median were classified as patients with low CAC, patients with CAC Score above the median as high CAC. In patients with high CAC compared to patients with low CAC we detected higher levels of TATc, platelet-derived MPs (PMPs), endothelial-derived MPs (EMPs) and MP-induced thrombin generation. Increased level of PMPs and MP-induced thrombin generation were independent predictors for the severity of CAC. In contrast, AVC Score did not differ between patients with high and low CAC and did neither correlate with MPs levels nor with MP-induced thrombin generation.

Conclusion

In patients with severe aortic valve stenosis MP-induced thrombin generation was independently associated with the severity of CAC but not AVC indicating different pathomechanisms involved in coronary artery and aortic valve calcification.     

Assaying Predatory Feeding Behaviors in Pristionchus and Other Nematodes

This protocol provides multiple methods for the analysis and quantification of predatory feeding behaviors in nematodes. Many nematode species including Pristionchus pacificus display complex behaviors, the most striking of which is the predation of other nematode larvae. However, as these behaviors are absent in the model organism Caenorhabditis elegans, they have thus far only recently been described in detail along with the development of reliable behavioral assays ¹. These predatory behaviors are dependent upon phenotypically plastic but fixed mouth morphs making the correct identification and categorization of these animals essential. In P. pacificus there are two mouth types, the stenostomatous and eurystomatous morphs ², with only the wide mouthed eurystomatous containing an extra tooth and being capable of killing other nematode larvae. Through the isolation of an abundance of size matched prey larvae and subsequent exposure to predatory nematodes, assays including both "corpse assays" and "bite assays" on correctly identified mouth morph nematodes are possible. These assays provide a means to rapidly quantify predation success rates and provide a detailed behavioral analysis of individual nematodes engaged in predatory feeding activities. In addition, with the use of a high-speed camera, visualization of changes in pharyngeal activity including tooth and pumping dynamics are also possible.