Proteolysis in septic shock patients: plasma peptidomic patterns are associated with mortality.

BACKGROUND: Uncontrolled proteolysis contributes to cell injury and organ dysfunction in animal models of circulatory shock. We investigated in humans the relationship between septic shock, proteolysis, and outcome. METHODS: Intensive care patients with septic shock (n=29) or sepsis (n=6) and non-hospitalised subjects (n=9) were recruited as part of the prospective observational trial 'ShockOmics' (ClinicalTrials.gov Identifier NCT02141607). A mass spectrometry-based approach was used to analyse the plasma peptidomes and the origin of circulating peptides from proteolysis in the enrolled subjects. RESULTS: Evidence of systemic proteolysis was indicated by a larger number of circulating peptides in septic shock patients, compared with septic patients and non-hospitalised healthy subjects. The peptide count and abundance in the septic shock patients were greater in patients who died (n=6) than in survivors (n=23), suggesting an association between magnitude of proteolysis and outcome. In silico analysis of the peptide sequences and of the sites of cleavage on the proteins of origin indicated a predominant role for serine proteases, such as chymotrypsin, and matrix metalloproteases in causing the observed proteolytic degradation. CONCLUSIONS: Systemic proteolysis is a novel fundamental pathological mechanism in septic shock. Plasma peptidomics is proposed as a new tool to monitor clinical trajectory in septic shock patients. CLINICAL TRIAL REGISTRATION: NCT02141607.

Immunological mechanisms involved in the human response to a dog dander allergoid.

BACKGROUND: Dog allergens are a common cause of allergic sensitisation and trigger respiratory symptoms worldwide. However, clinical evidence regarding dog immunotherapy is limited. Therefore, the aim of this study was to analyse the immunomodulatory properties of a new allergoid from dog dander, thereby deepening the understanding of the molecular mechanisms involved in the reestablishment of the tolerogenic response. METHODS: Three independent batches of dog dander native and allergoid allergen extracts were manufactured and characterised. Allergenic profiles were analysed by the identification of all dog allergens and quantification of the major allergens Can f 1 and Can f 5. The allergenicity profile of the allergoid was studied using biological potency and basophil activation tests. In vitro immunomodulatory parameters was evaluated as the capacity of the allergoid to induce IgG antibodies that block IgE binding to the allergen and cytokine promotion (IFN-γ, IL-4, IL-6, IL-10, IL-13, and TNF-α) in PBMCs from allergic donors. RESULTS: The presence of all dog allergens, including Can f 1 and Can f 5, was confirmed in both types of extracts. The new allergoid showed a low IgE binding capacity, which significantly affected the activation of effector cells, such as basophils. The IgG antibodies induced by the allergoid in rabbits blocked human IgE binding epitopes on the dog native extract and induced Th1 and Treg responses by increasing IFN-γ and IL-10 levels in PBMCs from allergic donors. CONCLUSION: This new dog dander allergoid containing Can f 1 and Can f 5 showed a low capacity to bind IgE and to activate basophils in dog allergic patients. Furthermore, it showed potent activation of Th1 mediators and induction of tolerance through Treg activation. This allergoid could offer a safer profile than the native extract and could be an effective immunotherapy treatment for dog allergic patients.

LC-MSMS identification of Arabidopsis thaliana heat-stable seed proteins: enriching for LEA-type proteins by acid treatment.

Protein identification in systems containing very highly abundant proteins is not always efficient and usually requires previous enrichment or fractionation steps in order to uncover minor proteins. In plant seeds, identification of late embryogenesis abundant (LEA) proteins is often masked by the presence of the large family of storage proteins. LEA-proteins are predicted to play a role in plant stress tolerance. They are highly hydrophilic proteins, generally heat-stable, and correlate with dehydration in seeds or vegetative tissues. In the present work, we analyze the protein composition of heat-stable Arabidopsis thaliana seed extracts after treatment with trichloroacetic acid (TCA). The composition of the proteins that precipitate and those that remain in solution in 3% TCA was analyzed by two different approaches: 1D SDS-PAGE coupled to LC-ESI-MSMS analysis and a gel-free protocol associated with LC-MALDI-MSMS. Our results indicate that treating total heat-soluble extracts with 3% TCA is an effective procedure to remove storage proteins by selective precipitation and this fractionation step provides a soluble fraction highly enriched in Lea-type proteins. The analysis and determination of protein identities in this acid-soluble fraction by MS technology is a suitable system for large-scale identification of Lea-proteins present in seeds.