Biomolecules capturing live bacteria from clinical samples.

Rapid phenotypic antimicrobial susceptibility testing (AST) requires the enrichment of live bacteria from patient samples, which is particularly challenging in the context of life-threatening bloodstream infections (BSIs) due to low bacterial titers. Over two decades, an extensive array of pathogen-specific biomolecules has been identified to capture live bacteria. The prevailing biomolecules are immune proteins of the complement system, antibodies, aptamers, phage proteins, and antimicrobial peptides. These biomolecules differ by their binder generation technologies and exhibit highly variable specificities, ranging from bacterial strains to most pathogenic bacteria. Here, we summarize how these diverse biomolecules were identified, list examples of successfully reported capture assays, and provide an outlook on the use of nanobodies raised against conserved surface-accessible proteins as promising biomolecules for pathogen capture.

Segmental pairs of dermal secretory cells release proteins into the hemolymph at the larval-pupal molt.

At each molt of Manduca, the large dermal secretory cells expel the protein contents of their vacuoles into the hemocoel. The constellation of proteins expelled at the last larval-pupal molt, however, differs qualitatively from those proteins released at earlier larval-larval molts. Secretory cells at the two stages not only have different lectin staining properties but also have different proteins that separate on two-dimensional gels. Numerous physiological changes accompany the termination of the last larval instar, including increased chitin synthesis, diminished oxygen delivery, and reduced humoral immunity. Secretion of trehalase that is essential for chitin synthesis and the release of hypoxia up-regulated protein to ameliorate oxygen deprivation help ensure normal transition from larva to pupa. Proteins released by dermal secretory cells at this last molt could supplement the diminished immune defenses mediated by fat body and hemocytes at the end of larval life. Additional immune defenses provided by dermal secretory cells could help ensure a safe transition during a period of increased vulnerability for the newly molted pupa with its soft, thin cuticle and reduced mobility.

Circulating Immune Proteins: Improving the Diagnosis and Clinical Outcome in Advanced Non-Small Cell Lung Cancer.

Immunotherapy has been proven a viable treatment option for non-small cell lung cancer (NSCLC) treatment in patients. However, some patients still do not benefit. Finding new predictive biomarkers for immunocheckpoint inhibitor (ICI) response will improve treatment management in the clinical routine. In this regard, liquid biopsy is a useful and noninvasive alternative to surgical biopsies. In the present study, we evaluated the potential diagnostic, prognostic, and predictive value of seven different soluble mediators involved in immunoregulation. Fifty-two plasma samples from advanced NSCLC treated in first-line with pembrolizumab at baseline (PRE) and at first response assessment (FR) were analyzed. In terms of diagnostic value, our results revealed that sFGL1, sGAL-3, and sGAL-1 allowed for optimal diagnostic efficacy for cancer patients. Additionally, the combination of sFGL1 and sGAL-3 significantly improved diagnostic accuracy. Regarding the predictive value to assess patients' immune response, sCD276 levels at PRE were significantly higher in patients without tumor response (p = 0.035). Moreover, we observed that high levels of sMICB at PRE were associated with absence of clinical benefit (pembrolizumab treatment less than 6 months) (p = 0.049), and high levels of sMICB and sGAL-3 at FR are also related to a lack of clinical benefit (p = 0.027 and p = 0.03, respectively). Finally, in relation to prognosis significance, at PRE and FR, sMICB levels above the 75th percentile are related to poor progression-free survival (PFS) (p = 0.013 and p = 0.023, respectively) and overall survival (OS) (p = 0.001 and p = 0.011, respectively). An increase in sGAL3 levels at FR was associated with worse PFS (p = 0.037). Interestingly, high sGAL-3 at PRE was independently associated with PFS and OS with a hazard ratio (HR) of 2.45 (95% CI 1.14-5.25; p = 0.021) and 4.915 (95% CI 1.89-12.73; p = 0.001). In conclusion, plasma levels of sFGL1, sGAL-3, and sGAL-1 could serve as diagnostic indicators and sMICB, sCD276, and sGAL3 were linked to outcomes, suggesting their potential in assessing NSCLC under pembrolizumab treatment. Our results highlight the value of employing soluble immune biomarkers in advanced lung cancer patients treated with pembrolizumab at first-line.

Insect Defense Proteins and Peptides.

The composition of insect hemolymph can change depending on many factors, e.g. access to nutrients, stress conditions, and current needs of the insect. In this chapter, insect immune-related polypeptides, which can be permanently or occasionally present in the hemolymph, are described. Their division into peptides or low-molecular weight proteins is not always determined by the length or secondary structure of a given molecule but also depends on the mode of action in insect immunity and, therefore, it is rather arbitrary. Antimicrobial peptides (AMPs) with their role in immunity, modes of action, and classification are presented in the chapter, followed by a short description of some examples: cecropins, moricins, defensins, proline- and glycine-rich peptides. Further, we will describe selected immune-related proteins that may participate in immune recognition, may possess direct antimicrobial properties, or can be involved in the modulation of insect immunity by both abiotic and biotic factors. We briefly cover Fibrinogen-Related Proteins (FREPs), Down Syndrome Cell Adhesion Molecules (Dscam), Hemolin, Lipophorins, Lysozyme, Insect Metalloproteinase Inhibitor (IMPI), and Heat Shock Proteins. The reader will obtain a partial picture presenting molecules participating in one of the most efficient immune strategies found in the animal world, which allow insects to inhabit all ecological land niches in the world.

Specific immunity proteomic profile of the skin mucus of Antarctic fish Chionodraco hamatus and Notothenia coriiceps.

The white-blooded Antarctic icefish is the only known vertebrate lacking oxygen-transporting haemoglobins. Fish skin mucus, as the first line of defence against pathogens, can reflect fish welfare. In this study, we analysed the skin mucus proteome profiles of the two Antarctic fish species, the white-blooded Antarctic icefish, Chionodraco hamatus, and the red-blooded Antarctic fish, Notothenia coriiceps, unfolding the different proteins by liquid chromatography coupled with tandem mass spectrometry isobaric tags for relative and absolute quantitation (iTRAQ) technology. Of the 4444 totally identified proteins, 227 differentially expressed proteins (DEPs) were found in the comparison between C. hamatus and N. coriiceps, of which 121 were upregulated and 106 were downregulated in the icefish. In the Kyoto Encyclopedia of Genes and Genomes pathway annotation, we found two pathways "Legionellosis" and "Complement and coagulation cascades" were significantly enriched, among of which innate immune candidate proteins such as C3, CASP1, ASC, F3 and C9 were significantly upregulated, suggesting their important roles in C. hamatus immune system. Additionally, the DEP protein-protein interaction network analysis and "Response to stress" GO category provided candidate biomarkers for deep understanding of the distinct immune response of the two Antarctic fish underlying the cold adaptation.

Role of immune microenvironment in gastrointestinal stromal tumours.

AIMS: The immune microenvironment is a prognostic factor for various malignancies. The significance of key players of this immune microenvironment, including tumour-infiltrating lymphocytes (TILs) and expression of programmed death-ligand 1 (PD-L1), indoleamine 2,3-dioxygenase (IDO) and tryptophanyl-tRNA synthetase (WARS) in gastrointestinal stromal tumours (GISTs) is largely unknown. METHODS AND RESULTS: Tissue microarrays were constructed from pathology files, 1996-2016. Immunohistochemistry for PD-L1, IDO and WARS was correlated with tumour size, mitoses and outcomes. TILs expressing CD3, CD4, CD8, FoxP3 and GBP5 were counted. A total of 129 GISTs were analysed. Mean patient age was 62.5 years; 52.0% were male. Tumour location included 89 stomach (69.0%), 33 small bowel (25.6%) and seven other (5.4%). Mean tumour size was 5.6 cm; mean mitoses were 7.2 per 50 high-power field. Nineteen patients (15.0%) developed disease progression, to abdominal wall (n = 8), liver (n = 6) and elsewhere (n = 5). Median progression-free survival was 56.6 months; five patients died of disease. PD-L1 was positive in 88 of 127 tumour samples (69.0%), 114 of 127 tumours were IDO-positive (89.8%) and 60 of 127 were positive for WARS (47.2%). PD-L1 was associated with increased size (P = 0.01), necrosis (P = 0.018) and mitoses (P = 0.006). Disease progression was not associated with PD-L1 (P = 0.44), IDO (P = 0.14) or WARS (P = 0.36) expression. PD-L1-positive GISTs with CD8+ or CD3+ TILs were significantly smaller than tumours with CD8+ or CD3+ TILs. CONCLUSIONS: PD-L1 expression was associated with increased size and mitoses. High CD8+ or CD3+ TIL counts were associated with decreased PD-L1/IDO+ GIST size. PD-L1 and IDO could be significant in GIST tumour biology, which invites consideration of immunotherapy as a potential treatment option.

CD3ζ-Mediated Signaling Protects Retinal Ganglion Cells in Glutamate Excitotoxicity of the Retina.

Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.

How Do ROS Induce NETosis? Oxidative DNA Damage, DNA Repair, and Chromatin Decondensation.

Neutrophil extracellular traps (NETs) are intricate, DNA-based, web-like structures adorned with cytotoxic proteins. They play a crucial role in antimicrobial defense but are also implicated in autoimmune diseases and tissue injury. The process of NET formation, known as NETosis, is a regulated cell death mechanism that involves the release of these structures and is unique to neutrophils. NETosis is heavily dependent on the production of reactive oxygen species (ROS), which can be generated either through NADPH oxidase (NOX) or mitochondrial pathways, leading to NOX-dependent or NOX-independent NETosis, respectively. Recent research has revealed an intricate interplay between ROS production, DNA repair, and NET formation in different contexts. UV radiation can trigger a combined process of NETosis and apoptosis, known as apoNETosis, driven by mitochondrial ROS and DNA repair. Similarly, in calcium ionophore-induced NETosis, both ROS and DNA repair are key components, but only play a partial role. In the case of bacterial infections, the early stages of DNA repair are pivotal. Interestingly, in serum-free conditions, spontaneous NETosis occurs through NOX-derived ROS, with early-stage DNA repair inhibition halting the process, while late-stage inhibition increases it. The intricate balance between DNA repair processes and ROS production appears to be a critical factor in regulating NET formation, with different pathways being activated depending on the nature of the stimulus. These findings not only deepen our understanding of the mechanisms behind NETosis but also suggest potential therapeutic targets for conditions where NETs contribute to disease pathology.

Functional analysis of Rehmannia glutinosa key LRR-RLKs during interaction of root exudates with Fusarium oxysporum reveals the roles of immune proteins in formation of replant disease.

Previous studies have indicated that some Rehmannia glutinosa Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are involved in the formation of replant disease. However, it remains unclear how the interaction of LRR-RLKs with a key factor, the interaction between root exudates and Fusarium oxysporum, results in formation of replant disease. In this study, the influences of root exudates, F. oxysporum and the interaction of these two factors on expression of nine R. glutinosa LRR-RLKs (RgLRRs) were analyzed. The resulting eight RgLRRs of them were highly expressed at the early stage, and rapidly declined at later stages under mixed treatment of root exudates and F. oxysporum. The functions of nine RgLRRs under root exudates, F. oxysporum and mixed treatment of root exudates and F. oxysporum were preliminarily analyzed using transient overexpression and RNAi experiments. The results showed that high expression of RgLRR19, RgLRR21, RgLRR23 and RgLRR29 could decrease the damage to root cells from the mixed treatment of root exudates and F. oxysporum, but the interference of these genes enhanced the damage levels of root cells. Based on this, stable transgenic R. glutinosa seedlings were acquired. Overexpression of RgLRR29 conferred resistance of R. glutinosa seedlings to root exudates, F. oxysporum and mixed treatment. These results indicated that the continuous proliferation of F. oxysporum supported by root exudates altered the expression patterns of RgLRRs in R. glutinosa, then disordered the growth and development of R. glutinosa, finally leading to the formation of replant disease.

Clinical Relevance of Soluble Forms of Immune Checkpoint Molecules sPD-1, sPD-L1, and sCTLA-4 in the Diagnosis and Prognosis of Ovarian Cancer.

It is crucial to find new diagnostic and prognostic biomarkers. A total of 80 patients were enrolled in the study. The study group consisted of 37 patients with epithelial ovarian cancer, and the control group consisted of 43 patients with benign ovarian cystic lesions. Three proteins involved in the immune response were studied: PD-1, PD-L1, and CTLA-4. The study material was serum and peritoneal fluid. The ROC curve was plotted, and the area under the curve was calculated to characterize the sensitivity and specificity of the studied parameters. Univariate and multivariate analyses were performed simultaneously using the Cox regression model. The cut-off level of CTLA-4 was 0.595 pg/mL, with the sensitivity and specificity of 70.3% and 90.7% (p = 0.000004). Unfavorable prognostic factors determined in serum were: PD-L1 (for PFS: HR 1.18, 95% CI 1.11-1.21, p = 0.016; for OS: HR 1.17, 95% CI 1.14-1.19, p = 0.048) and PD-1 (for PFS: HR 1.01, 95% CI 0.91-1.06, p = 0.035). Unfavorable prognostic factors determined in peritoneal fluid were: PD-L1 (for PFS: HR 1.08, 95% CI 1.01-1.11, p = 0.049; for OS: HR 1.14, 95% CI 1.10-1.17, p = 0.045) and PD-1 (for PFS: HR 1.21, 95% CI 1.19-1.26, p = 0.044). We conclude that CTLA-4 should be considered as a potential biomarker in the diagnosis of ovarian cancer. PD-L1 and PD-1 concentrations are unfavorable prognostic factors for ovarian cancer.

Clinical Significance of BTLA, CD27, CD70, CD28 and CD80 as Diagnostic and Prognostic Markers in Ovarian Cancer.

It is very important to find new diagnostic and prognostic biomarkers. A total of 79 patients were enrolled in the study. The study group consisted of 37 patients with epithelial ovarian cancer, and the control group consisted of 42 patients with benign ovarian lesions. Five proteins involved in the immune response were studied: BTLA, CD27, CD70, CD28, CD80. The study material was serum and peritoneal fluid. The ROC curve was plotted, and the area under the curve was calculated to characterize the sensitivity and specificity of the studied parameters. Univariate and multivariate analyses were performed simultaneously using the Cox regression model. The cut-off level of CD27 was 120.6 pg/mL, with the sensitivity and specificity of 66 and 84% (p = 0.014). Unfavorable prognostic factors determined in serum were: CD27 (for PFS: HR 1.26, 95% CI 1.21-1.29, p = 0.047; for OS: HR 1.20, 95% CI 1.15-1.22, p = 0.014). Unfavorable prognostic factors determined in peritoneal fluid were: BTLA (for OS: HR 1.26, 95% CI 1.25-1.31, p = 0.033). We conclude that CD27 should be considered as a potential biomarker in the diagnosis of ovarian cancer. BTLA and CD27 are unfavorable prognostic factors for ovarian cancer.

iTRAQ-based Quantitative Proteomics Analysis Identifies Host Pathways Modulated during Toxoplasma gondii Infection in Swine.

Toxoplasma gondii is a leading cause of foodborne illness and consumption of undercooked pig meat is a major risk factor for acquiring toxoplasmosis, which causes a substantial burden on society. Here, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify cellular proteins and pathways altered during T. gondii infection in pigs. We also used parallel reaction monitoring-based LC-MS/MS to verify the levels of protein expression of infected spleens and mesenteric lymph nodes (MLNs). At 6 days post-infection (dpi), 156, 391, 170, 292, and 200 differentially expressed proteins (DEPs) were detected in the brain, liver, lung, MLNs and spleen, respectively. At 18 dpi, 339, 351, 483, 388, and 303 DEPs were detected in the brain, liver, lung, MLNs and spleen, respectively. Although proteins involved in immune responses were upregulated in all infected tissues, protein expression signature in infected livers was dominated by downregulation of the metabolic processes. By weighted gene co-expression network analysis, we could further show that all proteins were clustered into 25 co-expression modules and that the pink module significantly correlated with the infection status. We also identified 163 potential anti-T. gondii proteins (PATPs) and provided evidence that two PATPs (HSP70.2 and PDIA3) can reduce T. gondii burden in porcine macrophages in vitro. This comprehensive proteomics analysis reveals new facets in the pathogenesis of T. gondii infection and identifies key proteins that may contribute to the pig's defense against this infection.

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins.

Glycans and glycan binding proteins (GBPs or lectins) are essential components in almost every aspect of immunology. Investigations of the interactions between glycans and GBPs have greatly advanced our understanding of the molecular basis of these fundamental immunological processes. In order to better study the glycan-GBP interactions, microscope glass slide-based glycan microarrays were conceived and proved to be an incredibly useful and successful tool. A variety of methods have been developed to better present the glycans so that they mimic natural presentations. Breakthroughs in chemical biology approaches have also made available glycans with sophisticated structures that were considered practically impossible just a few decade ago. Glycan microarrays provide a wealth of valuable information in immunological studies. They allow for discovery of detailed glycan binding preferences or novel binding epitopes of known endogenous immune receptors, which can potentially lead to the discovery of natural ligands that carry the glycans. Glycan microarrays also serve as a platform to discover new GBPs that are vital to the process of infection and invasion by microorganisms. This review summarizes the construction strategies and the immunological applications of glycan microarrays, particularly focused on those with the most comprehensive sets of glycan structures. We also review new methods and technologies that have evolved. We believe that glycan microarrays will continue to benefit the growing research community with various interests in the field of immunology.

The Vaginal Community State Types Microbiome-Immune Network as Key Factor for Bacterial Vaginosis and Aerobic Vaginitis.

Regarding bacterial vaginosis (BV), the relevance of the vaginal microbiota to the women's health fulfills a key role, but knowledge gaps regarding aerobic vaginitis (AV) exist. This study aims to characterize vaginal microbiome and its relationship with the local immune mediators, providing an opportunity to define the link between vaginal commensal microorganisms and opportunistic pathogens in the relation of a given vaginal community state type (CST). A total of 90 vaginal samples from Caucasian asymptomatic women of reproductive age (18-40 years) attending the yearly examination and not reporting any vaginal complaints were retrospectively evaluated for microbiome assessment and immune factor dosage. The samples were tested by the Ion Torrent PGM and the Luminex Bio-Plex technologies for the analysis of microbiome and immune factors, respectively. In our study, the CST classification together with the local immune response profiling represented a good predictive indicator of the vaginal health, suggesting that the predominance of a specific Lactobacillus and its relative abundance are pivotal elements to maintain a physiologic status. A vaginal colonization from Bifidobacterium may absolve a protective role similar to that of Lactobacillus, corresponding to a newly identified CST, although studies are needed to better clarify its clinical significance. Moreover, within each CST, a different pattern of inflammation is activated and orchestrated both by the dominant Lactobacillus spp. and by specific non-Lactobacillus bacteria and can give insights into the pathogenic mechanisms. In conclusion, this study contributes to the characterization of vaginal dysbiosis, reshaping this concept by taking into consideration the CST profiling, local immune marker, and immune-microbial network.

Comparative proteomics analysis of human and ruminant milk serum reveals variation in protection and nutrition.

In present study, 198, 169, 213 and 128 proteins were identified and quantified in human, cow, goat and yak milk serum respectively by using proteomics techniques. Large variations were observed between human and ruminant milk proteins. Human milk contained higher concentration of mucosal immune response, complement proteins and regulators. The concentration of bactericidal proteins were relatively higher in ruminants milk. Human milk exclusively expressed proteins important for delivery or utilization of nutrients. Peptidase inhibitors prevent the bioactive proteins/peptides in human milk from gastral-intestinal digestion. Protein composition among ruminants milk was similar but with variations. Goat milk contained high level of complement proteins but low level of corresponding regulators. In addition, the acute phase proteins were significantly higher in goat milk. Of note, osteopontin in yak milk was enriched, which could offer an alternative source for producing osteopontin and revealed possible nutritional value of yak milk.

A New High Hydrostatic Pressure Process to Assure the Microbial Safety of Human Milk While Preserving the Biological Activity of Its Main Components.

Background: The main process used to pasteurize human milk is the low-temperature, long-time Holder method. More recently, the high-temperature, short-time method has been investigated. Both processes lead to the appropriate inactivation of vegetative bacterial forms but are ineffective against bacterial spores. Research Aims/Questions: We aimed to accomplish two main objectives: inactivation of all pathogens, including spores; and preservation of the activity of milk components. Design/Methods: Recently, a novel high-hydrostatic pressure process has been developed by HPBioTECH. Using the same raw human milk samples, we compared the effects of this method with those of the Holder method on vegetative and spore forms of pathogens and on bioactive components (lipase activity, immunoproteins). Results: Two main microbial strains were selected: Staphylococcus aureus (as a reference for vegetative forms) and Bacillus cereus (as a reference for spores). Use of the high-hydrostatic pressure process led to microbial decontamination of 6 log for both S. aureus and B. cereus. Additionally, the bioactivity of the main components of human milk was preserved, with activities of lipase, α-lactalbumin, casein, lysozyme, lactoferrin, and sIgA of ~80, 96-99, 98-100, 95-100, 93-97, and 63-64%, respectively. Conclusions: Use of this novel high-hydrostatic pressure process to generate microbiologically safe human milk may provide important benefits for preterm infants, including improved assimilation of human milk (leading increased weight gain) and improved resistance to infections. Because 10% of all human milk collected is contaminated by B. cereus, use of this method will also prevent waste.

T-cell receptors modify neuronal function in the central nervous system.

Recent published findings have shown that many proteins discovered in the immune system and residing on immune cells with well established immune-related functions are also found in neurons of the central nervous system. These studies have uncovered a rich variety of neuronal functions attributed to these immune proteins. This review will focus on two key interacting protein complexes that previously were known for adaptive immune reactions, the major histocompatability complex and the T-cell receptor complex. We will review where these immune proteins are expressed in the CNS and their neuronal function.