Influence of blood collection methods and long-term plasma storage on quorum-sensing peptide stability

Finding adequate biomarkers for rapid and accurate disease detection, prognosis, and therapy is increasingly important. Quorum-sensing peptides are herein a new emerging group, produced by bacteria, fungi, protozoa, and viruses, with blood being the most straightforward sample type to detect/quantitate them. However, detailed information about suitable blood sample collection methods and storage conditions for measuring these quorum-sensing peptides hampers further clinical research and development. Here, we first tested the time-dependent stability of a set of chemically diverse quorum-sensing peptides, spiked in blood at different temperatures (4, 21, and 37 °C) in four different ethylenediamine tetraacetic acid (EDTA)-containing plasma tubes (with different protein-stabilizing additives) over a period of up to 7.5 h. Next, we determined the storage stability of these quorum-sensing peptides in plasma at different temperatures (4, −35, and −80 °C). UPLC/MS–MS was used to selectively detect and quantify the spiked quorum-sensing peptides. The results of this study indicate that a cost-effective tube, designed for traditional proteomics and stored at 4 °C, is the preferred collection condition when quorum-sensing peptides need to be detected/quantified in human plasma. When the tubes are handled at room temperature (21 °C), a more specialized tube is required. Long-term storage of plasma samples, even under low-temperature conditions (−80 °C), indicates rapid degradation of certain quorum-sensing peptides

An unusual presentation of a case of human psittacosis

Background: Chlamydia psittaci is a gram-negative, obligate intracellular organism. Birds are the main reservoir, but also non-avian domestic animals and humans can be infected. In humans it mostly causes respiratory infections due to occupational exposure with varying severity. Sensitive and specific diagnostic tests are needed to define psittacosis in humans as these tests also allow rapid tracing of the animal source. However, diagnosis in humans is often based on time-consuming culture techniques and antibody detection assays as in many countries, the existing molecular diagnostic tests for psittacosis are not reimbursed by the public health insurance. Case presentation: An 82-year old female was referred to the hospital with a non-productive cough since four weeks and since one week fever up to 39 degrees C, myalgia, generalized skin rash, acral edema and generalized weakness under treatment with moxifloxacin. Blood analysis showed signs of inflammation with mild eosinophilia. Chest CT showed multiple peripheral ground glass opacities with consolidation in both lungs. Pulmonary function testing only showed a mild decrease in diffusion capacity. Viral and bacterial serology were negative. As the patient kept a pet parakeet for over ten years, a nested PCR for C. psittaci was performed on a nasopharyngeal swab of the patient and on feces of the parakeet. Both returned positive for the same genotype. Genotyping was performed by a genotype-specific real-time PCR. The patient fully recovered after a ten-day course of azithromycin. Conclusion: Due to non-specific signs during psittacosis, early detection of the infection and differentiation from hypersensitivity pneumonitis can be challenging. Culture and antibody titers for C. psittaci have a lower sensitivity than PCR-testing due to several factors. We present a case of human psittacosis (presenting as pneumonia) with diagnosis based on clinical findings confirmed by means of nested PCR. This case suggests the added value of PCR in suspect cases despite negative serology. Our current paper underlines the need for a broader implementation of PCR for early diagnosis of human psittacosis and thus early initiation of correct antibiotic treatment with reduction of morbidity and mortality

From immune senescence and insulin resistance to obesity and sarcopenia: iNKT cell as bridge?

In the physiological process of aging, different major players are involved. Sarcopenia, defined as a critical loss of muscle mass and function, an increase of the visceral fat proportion, insulin resistance and immune senescence are all contributing to the disability and mortality in older adults. The interactions between these communicating processes and the molecular mechanisms underlying them are poorly understood but increasing evidence is pointing towards inflammatory cytokines as common link. Adipokines and myokines are important in the pathogenesis of sarcopenia, obesity and insulin resistance, with IFNg, TNFa, IL-6, IL-2, IL-8, MCP-1 and IL-15 as known cytokines taking part herein. Despite this knowledge, the role of immune senescence and of possible key ageing immune cells in this network , is still an unresolved question. We speculate that part of the answer can be found in a relatively recent discovered leukocyte type: the invariant natural killer T-cell (iNKT). iNKT cells are a regulatory type of T-cells with innate-like properties that recognize glycolipids in the context of the non-classical MHC molecule CD1d. Our hypothesis of a pivotal role for iNKT cells in sarcopenia, visceral obesity, insulin resistance and immune senescence is based on the following insights: (i) iNKT cells are very potent and flexible cytokine-producers that secrete large amounts of Th1 and/or Th2 cytokines depending on the physiological context; (ii) iNKT cells quantitatively and qualitatively change with aging, e.g. the peripheral iNKT cell amount decreases with age; (iii) iNKT cells play a crucial role in obesity-induced insulin resistance; (iv) impaired mitochondrial autophagy in sarcopenia is related to dysfunctional immune system; (v) there is a bidirectional crosstalk between sarcopenia and insulin resistance. This proposed link will be explored by investigating how selected endogenous and exogenous glycolipids affect muscle mass and function. As such, our findings will contribute to an improved pathophysiological understanding of sarcopenia with diagnostic and therapeutic clinical applications

A computational high-throughput screening approach of iNKT-agonists: a novel tool to find optimized iNKT cell ligands

Depending on the environment and the activating glycolipids, iNKT cells are known to induce T-helper 1 and/or T-helper 2 cytokines. This highly versatile nature makes these innate-like cells very interesting targets for immunomodulation. As many pathologies as well as physiological ageing are associated with altered immune responses, iNKT cells could play a role in new therapies. Many analogs of the glycolipid alpha-galactosylceramide (a-GalCer) are known to activate iNKT-cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and/or Th2 cytokines. The design of iNKT cell ligands with selective Th1 and Th2 properties requires refined structural insights. Therefore, the chemical space of 333 currently known iNKT activators, including several newly tested analogs, was visualized by more than 3000 chemical descriptors which were calculated for each individual analog. The immunological responses consisted of four different cytokines in five different test-systems. With these two information-sets, structure-activity models were developed using a system biology computational approach. We present highly sensitive and specific predictive models that can be further exploited as high-throughput instruments to in-silico screen potential glycolipids, thereby reducing the attrition rate

An in silico approach for modelling T-helper polarizing iNKT cell agonists

Many analogues of the glycolipid alpha-galactosylceramide (α-GalCer) are known to activate iNKT cells through their interaction with CD1d-expressing antigen-presenting cells, inducing the release of Th1 and Th2 cytokines. Because of iNKT cell involvement and associated Th1/Th2 cytokine changes in a broad spectrum of human diseases, the design of iNKT cell ligands with selective Th1 and Th2 properties has been the subject of extensive research. This search for novel iNKT cell ligands requires refined structural insights. Here we will visualize the chemical space of 333 currently known iNKT cell activators, including several newly tested analogues, by more than 3000 chemical descriptors which were calculated for each individual analogue. To evaluate the immunological responses we analyzed five different cytokines in five different test-systems. We linked the chemical space to the immunological space using a system biology computational approach resulting in highly sensitive and specific predictive models. Moreover, these models correspond with the current insights of iNKT cell activation by α-GalCer analogues, explaining the Th1 and Th2 biased responses, downstream of iNKT cell activation. We anticipate that such models will be of great value for the future design of iNKT cell agonists

PapRIV, a BV-2 microglial cell activating quorum sensing peptide

Quorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction

The paradox of scored tablets: a cost-saving risk

One of the cornerstones of pharmacotherapy is the proper dose of medicine, which should ideally be tailored to the individual patient. However, even if clinically possible, this is economically not feasible as a too large number of different dosage strengths would be required. Therefore, a balance is required between the patient's benefit/risk and the cost to the individual and society on the other hand. Scored or splitted tablets were, and still are, often used strategies to these opposite interests, enabling more dose-flexibility, but also at the same time increasing the dose-variability as a consequence of the breaking process. The question of how to deal with this paradox was investigated by exploring the prevalence and classification of scored tablets as well as the cost-benefits. A strategy for clinical pharmacologists is presented to improve the outcome of this paradox

Peptidomics : LC-MS operational parameters do matter

The sensitive and specific detection of peptides at low levels in biofluids is critical to increase the lab-to-human translation of peptidomic research. An interesting group of peptides with increasing evidence for involvement in human diseases are quorum sensing peptides. To obtain more reliable conclusions on peptide measurands in biofluids, a selection of often neglected parts of the analytical process using LC-MS were investigated, with novel approaches recommended for each part. Quorum sensing peptides were used as the main model-peptides. The peptidomic parts investigated and discussed here are: 1. Sample collection: obtaining and storing biofluids such as plasma/serum, feces or saliva from mice and humans up to the laboratory handling (sample preparation) should ensure no degradation/metabolization of the peptide measurand (yielding false negatives) or of proteins (yielding false positives). 2. Sample preparation: to remove interfering compounds as well as to release peptides from the adsorbing matrix-components and to preconcentrate them; this is a crucial step which should assure analytical stability and adsorption-minimization. 3. Chromatography: not only the separation power and orthogonality of the complementary systems is critical, but a careful characterisation of the gradient-system, such as the starting conditions, need to be addressed as well because of worsened detection limits (yielding false negatives) or carry-over (false positives). 4. MS detection: operational parameters such as duty cycle characteristics applied are critical in obtaining low-level, reliable results. Our work addresses aQbD-approached solutions to these challenges, encompassing sample stabilization measures, a suitable peptide anti-adsorption tool, judicious choice of injection solvent versus gradient system and optimal duty cycle parameters. Our recommendations will improve the peptidomics bio-analytics of not only quorum sensing peptides, but can also be of value for other measurands at low concentrations