Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome

Background

Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form.

Methodology and Principal Findings

The mRNA abundance of cytokines (IL-1α, IL-1β, IL-8, IL-10, TNF-α, TGF-β) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1β, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury.

Conclusion

The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.     

Study of solid-phase synthesis and purification strategies for the preparation of polyglutamine peptides

Many neurodegenerative diseases are related to an abnormal expansion of the CAG trinucleotide that produces polyglutamine segments in several proteins. However, the pathogenesis of these neurodegenerative states is not yet well understood. Thus, to evaluate the molecular mechanisms leading to those diseases, suitable research tools such as synthetic polyglutamine peptides are required. The synthesis and purification of such peptides are usually difficult because of poor solubility, which leads to low coupling and/or deblocking reactivity. After exploring many synthesis, solubilization and purification approaches, a protocol allowing the production of polyglutamines in good yield and high purity was developed. With this protocol, peptides of 10-30 glutamine residues were synthesized using a linear solid-phase strategy combined with a maximal side-chain protection scheme using fluorenylmethyloxycarbonyl (Fmoc) chemistry. After cleavage of the peptide from the polymeric support, the crude material was treated with glacial acetic acid and lyophilized. This treatment significantly improved the solubility of the polyglutamine peptides thus allowing their dissolution in aqueous conditions and purification through reverse-phase high performance liquid chromatography. These solubilization and purification conditions led to the formation of N-pyroglutamyl peptide derivatives that were easily isolated. These N-pyroglutamylated compounds also appear as useful research tools because data from the literature suggest that N-terminal modification of polyglutamine segments might play a role in their pathogenic properties.     

Prevention and treatment of enteric viral infections: possible benefits of probiotic bacteria

The structure and function of the intestinal epithelium is briefly described, with the principal mechanisms involved in diarrhea. Human enteric viruses and probiotics are presented. We then review how probiotic bacteria could interfere with virus-induced pathology, we present our own view and describe specific interactions that would be valuable targets for future studies.     

Heme acquisition by Shu1 requires Nbr1 and proteins of the ESCRT complex in Schizosaccharomyces pombe

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4⁺ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA₄‐tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1⁺, ESCRT‐0 hse1⁺ or ESCRT‐I sst6⁺ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin‐treated cells, Shu1‐Nbr1 and Shu1‐Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin‐treated cells harboring a deletion for one of the following genes: ubi4⁺, nbr1⁺, hse1⁺ and sst6⁺. Together, these results reveal that hemin‐mediated vacuolar targeting of Shu1 requires Ubi4‐dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.     

Heterozygous mutations in TREX1 cause familial chilblain lupus and dominant Aicardi-Goutieres syndrome

TREX1 constitutes the major 3'-->5' DNA exonuclease activity measured in mammalian cells. Recently, biallelic mutations in TREX1 have been shown to cause Aicardi-Goutieres syndrome at the AGS1 locus. Interestingly, Aicardi-Goutieres syndrome shows overlap with systemic lupus erythematosus at both clinical and pathological levels. Here, we report a heterozygous TREX1 mutation causing familial chilblain lupus. Additionally, we describe a de novo heterozygous mutation, affecting a critical catalytic residue in TREX1, that results in typical Aicardi-Goutieres syndrome.     

Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.