Peripheral blood mononuclear cell transcriptome profile in a clinical trial with subcutaneous, grass pollen allergoid immunotherapy.

INTRODUCTION: Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment in allergic airway diseases. Underlying immunological mechanisms and candidate biomarkers, which may be translated into predictive/surrogate measures of clinical efficacy, remain an active area of research. The aim of this study was to evaluate Pollinex Quattro (PQ) Grass AIT induced immunomodulatory mechanisms, based on transcriptome profiling of peripheral blood mononuclear cells. METHODS: 119 subjects with grass pollen induced seasonal allergic rhinitis (SAR) were randomized in a 2:2:1:1 ratio to receive a cumulative dose of PQ Grass as a conventional or extended pre-seasonal regimen, placebo, or placebo with MicroCrystalline Tyrosine. Gene expression analysis was an exploratory endpoint evaluated in a subgroup of 30 subjects randomly selected from the four treatment arms. Samples were collected at three time points: screening (baseline), before the start of the grass pollen season and at the end of the season. This study was funded by the manufacturer of PQ. RESULTS: Transcriptome analysis demonstrated that the most significant changes in gene expression, for both treatment regimens, were at the end of the grass pollen season, with the main Th1 candidate molecules (IL-12A, IFNγ) upregulated and Th2 signature cytokines downregulated (IL-4, IL-13, IL-9) (p < .05). Canonical pathways analysis demonstrated Th1, Th2, Th17 and IL-17 as the most significantly enriched pathways based on absolute value of activation z-score (IzI score ≥ 2, p < .05). Upstream regulator analysis showed pronounced inhibition of pro-inflammatory allergic molecules IgE, IL-17A, IL-17F, IL-25 (IL-17E) (IzI score ≥ 2, FDR < 0.05) and activation of pro-tolerogenic molecules IL-12A, IL-27, IL-35 (EBI3) at the end of the grass pollen season. CONCLUSION: Peripheral blood mononuclear cells transcriptome profile showed an inhibition of Th2, Th17 pro-inflammatory allergic responses and immune deviation towards Th1 responses. PQ Grass extended regimen exhibited a superior mechanistic efficacy profile in comparison with PQ conventional regimen.

Nurses' autonomy in sleep management improves patients' sleep quality: A cross-sectional study.

BACKGROUND: The current literature indicates that intensive care (ICU) patients' sleep quality is generally poor, which is associated with serious physical and psychological consequences. AIMS AND OBJECTIVES: To describe the practices nurses use to provide good-quality sleep to adult ICU patients and assess nurses' perceptions of patients' sleep quality and nurses' professional autonomy in sleep management. DESIGN: A descriptive-correlational, cross-sectional study. METHODS: A total of 232 ICU nurses from four hospitals in Poland were recruited. Data were collected between May and August 2019 using a previously developed questionnaire and analysed using descriptive statistics and non-parametric tests. RESULTS: A total of 119 nurses took part in the study (response rate: 51%). On average, nurses rated patients' sleep quality as moderate (4.44 ± 2.23, scale 0-10). Most of the respondents (95.8%) said they did not use any sleep protocol. Various strategies to improve patients' sleep were used sporadically (2.64 ± 1.55, scale 1-5). The use of sleep quality assessment methods was positively correlated with patients' sleep quality (rho = 0.22, P = .02). Nurses' professional autonomy regarding sleep management was assessed as average (4.34 ± 2.43, scale 0-10) and was correlated with the patients' sleep quality (rho = 0.25, P < .01). Nurses who rated their autonomy in patients' sleep management more highly (rho = 0.29, P < .01) and more often influenced patients' sleep decisions (rho = 0.24, P < .01) used more methods to improve patients' sleep. CONCLUSIONS: Strengthening the professional autonomy of ICU nurses and creating a reliable sleep assessment and improvement tool, which would describe strategies nurses can implement independently could increase sleep quality among ICU patients. RELEVANCE TO CLINICAL PRACTICE: Addressing organizational problems, which hamper the patients' sleep management by ICU nurses could result in using more strategies to provide good-quality sleep to ICU patients. There is a need for clinical guidelines regarding patients' sleep management to help educate and guide nurses how to independently use sleep improvement methods.

Vitamin D Binding Protein Isoforms and Apolipoprotein E in Cerebrospinal Fluid as Prognostic Biomarkers of Multiple Sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a multifactorial autoimmune disease of the central nervous system with a heterogeneous and unpredictable course. To date there are no prognostic biomarkers even if they would be extremely useful for early patient intervention with personalized therapies. In this context, the analysis of inter-individual differences in cerebrospinal fluid (CSF) proteome may lead to the discovery of biological markers that are able to distinguish the various clinical forms at diagnosis. METHODS: To this aim, a two dimensional electrophoresis (2-DE) study was carried out on individual CSF samples from 24 untreated women who underwent lumbar puncture (LP) for suspected MS. The patients were clinically monitored for 5 years and then classified according to the degree of disease aggressiveness and the disease-modifying therapies prescribed during follow up. RESULTS: The hierarchical cluster analysis of 2-DE dataset revealed three protein spots which were identified by means of mass spectrometry as Apolipoprotein E (ApoE) and two isoforms of vitamin D binding protein (DBP). These three protein spots enabled us to subdivide the patients into subgroups correlated with clinical classification (MS aggressive forms identification: 80%). In particular, we observed an opposite trend of values for the two protein spots corresponding to different DBP isoforms suggesting a role of a post-translational modification rather than the total protein content in patient categorization. CONCLUSIONS: These findings proved to be very interesting and innovative and may be developed as new candidate prognostic biomarkers of MS aggressiveness, if confirmed.

Molecular and immunological characterization of three strains of Anaplasma marginale grown in cultured tick cells.

Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines.

Use of Percoll gradients to purify Anaplasma marginale (Rickettsiales: Anaplasmataceae) from tick cell cultures.

Anaplasma marginale (Rickettsiales: Anaplasmataceae) is an obligate intracellular bacterium that multiplies exclusively within membrane-bound vacuoles in the cytoplasm of host cells. A number of A. marginale isolates can be propagated in the Ixodes scapularis IDE8 tick cell line, which provides a reliable source of antigens for a wide variety of studies. However, because of its intracellular nature, separation of bacteria from host cell materials remains an important constraint for researchers. In the present study, we evaluated the use of Percoll gradients for purification of two Brazilian strains of A. marginale grown in IDE8 tick cells. The purified A. marginale monitored in Giemsa-stained smears contained only minimal amounts of IDE8 cell stroma. The total protein yields were 1.2mg and 1.7mg, while the DNA titers quantified with real-time PCR were 6.4×10(9) for UFMG1 and 4.87×10(9) for UFMG2 copies in the purified material, respectively. Additionally, we confirmed the viability of purified bacteria by infecting tick cells after being freshly purified and after retrieval from long-term storage. Importantly, the viability of the organisms is preserved after use of this separation method, and therefore the purified organisms can be used in enzymatic assays and other research approaches where live organisms would be preferred.

In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.

Functional and immunological relevance of Anaplasma marginale major surface protein 1a sequence and structural analysis.

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5'-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5'-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.

The activity of aminoacyl-tRNA synthetase-interacting multi-functional protein 1 (AIMP1) on endothelial cells is mediated by the assembly of a cytoskeletal protein complex.

AIMP1 was first found as a factor associated with the aminoacyl-tRNA synthetase (ARS) complex. However, it is also secreted and acts on different target cells such as endothelial cells, macrophages, and fibroblasts as an extracellular regulator, respectively, of angiogenesis, inflammatory responses and dermal regeneration. AIMP1 has also been reported to suppress in vivo tumor growth. In this study, we investigated the signaling pathways activated by exogenous AIMP1 in an in vitro endothelial model. AIMP1 decreases EC viability through an α5ß1 integrin-dependent mechanism and inhibits cell adhesion, is internalized and shows an asymmetric pattern of distribution and accumulation in cell protrusions. Experiments of affinity purification, pull down, and co-immunoprecipitation showed that AIMP1 interacts with four cytoskeletal proteins (filamin-A, α-tubulin, vinculin, and cingulin). α-Tubulin also gets phosphorylated upon cell treatment with AIMP1 and colocalization between AIMP1 and filamin-A as well as between AIMP1 and cingulin was observed through immunofluorescence assays. In this work, we propose that AIMP1 effect on EC adhesion is mediated by the assembly of a cytoskeletal protein complex on the cytosolic face of the cell membrane which could regulate cellular architecture maintenance and remodeling. Moreover, this activity is able to indirectly influence cell viability.

Acute-phase proteins investigation based on lectins affinity capture prior to 2-DE separation: application to serum from multiple sclerosis patients.

Plasma acute-phase proteins (APPs) glyco-isoforms are important biomarkers of inflammatory processes such as those occurring in multiple sclerosis (MS). Specific analysis of these proteins is often hampered by sample biochemical complexity. The aim of our study was to set up a method to accurately visualize, identify and quantify APPs glyco-isoforms in human serum. An enrichment strategy based on affinity chromatography using the carbohydrate-binding proteins concanavalin A (ConA) and erythrina cristagalli lectin (ECL) was applied to pooled serum samples from 15 patients and 9 healthy individuals. Image analysis of 2-DE detected 30 spots with a fold change higher than 1.5. A total of 14 were statistically significant (p value<0.05): 7 up-regulated and 7 down-regulated in MS samples. ESI LC-Nanospray IT mass spectrometry analysis confirmed that all of them were APPs isoforms supporting the idea that the accurate analysis of differential glycosylation profiles in these biomarkers is instrumental to distinguish between MS patients and healthy subjects. Additionally, overlaps in ConA/ECL maps protein patterns suggest how the used lectins are able to bind sugars harbored by the same oligosaccharide structure. Among identified proteins, the presence of complex and/or hybrid type N-linked sugar structures is well known. Performing galectin-3 binding and Western blotting, we were able to demonstrate a correlation between hybrid type glyco-isoforms of ß-haptoglobin and MS. In conclusion, although the patho-physiological role of the identified species still remains unclear and further validations are needed, these findings may have a relevant impact on disease-specific marker identification approaches.