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Sigma-class glutathione transferase (GST) proteins with dual GST and prostaglandin synthase (PGS) activities play a crucial role in the establishment of Clonorchis sinensis infection. Herein, we analyzed the structural and enzymatic properties of sigma-class GST (CsGST-σ) proteins to obtain insight into their antioxidant and immunomodulatory functions in comparison with mu-class GST (CsGST-μ) proteins. CsGST-σ proteins conserved characteristic structures, which had been described in mammalian hematopoietic prostaglandin D2 synthases. Recombinant forms of these CsGST-σ and CsGST-μ proteins expressed in Escherichia coli exhibited considerable degrees of GST and PGS activities with substantially different specific activities. All recombinant proteins displayed higher affinities toward prostaglandin H2 (PGS substrate; average Km of 30.7 and 3.0 μm for prostaglandin D2 [PGDS] and E2 synthase [PGES], respectively) than those toward CDNB (GST substrate; average Km of 1,205.1 μm). Furthermore, the catalytic efficiency (Kcat/Km) of the PGDS/PGES activity was higher than that of GST activity (average Kcat/Km of 3.1, 0.7, and 7.0×10-3 s-1μm-1 for PGDS, PGES, and GST, respectively). Our data strongly suggest that the C. sinensis sigma- and mu-class GST proteins are deeply involved in regulating host immune responses by generating PGD2 and PGE2 in addition to their roles in general detoxification.

Background: Cow's milk allergy (CMA) is the most common IgE-mediated food allergy and Bos d 5 is the major allergen in cow's milk proteins. More than 60% of the patients with CMA are sensitized to this protein., Methods and Results: A recombinant protein, encoded by a synthetic gene and consisting of reassembled Bos d 5 fragments, was expressed in E. coli strain BL21 (DE3) cells and purified to homogeneity. The B5M lacked relevant IgE-reactivity and allergenic activity compared with Bos d 5 in dot-blot and basophil activation assays. T-cell proliferation experiments demonstrated that B5M preserved the main T cell epitopes of Bos d 5. Immunization of rabbits with B5M induced protective IgG antibodies that blocked the binding of patients' IgE antibodies to the wild-type allergen and inhibited the degranulation of basophils induced by Bos d 5., Conclusion: Thus, we developed a new strategy, which was based on rational molecular reassembly for allergen-specific immunotherapy (AIT) of CMA and food allergy., (© 2021 S. Karger AG, Basel.)

Background: Furry animals are an important source of indoor allergens. Diagnosis of allergy to small pets such as guinea-pigs still relies on animal dander extracts which do not allow to define the primary sensitization source., Objective: To identify major guinea-pig allergens and to evaluate their potential as marker allergens for in vitro IgE-diagnosis in comparison with dander extracts., Methods: A group of patients allergic to guinea-pig (n = 29) and a group of patients allergic to cat and dog (n = 30) were recruited for the study. A panel of four guinea-pig lipocalin allergens was expressed as recombinant proteins in E. coli. Specific IgE were quantified by ImmunoCAP and ELISA., Results: The combination of 4 guinea-pig lipocalin allergens, including 2 new lipocalins, Cav p 1.0201 and Cav p 6.0101, and the previously characterized lipocalins Cav p 2 and Cav p 3, enabled the identification of 90% of all patients allergic to guinea-pig. The vast majority had specific IgE to Cav p 1 (83%). Cav p 6 shares 54% sequence identity with Fel d 4 and Can f 6 and was found to be IgE-cross-reactive with these allergens. In the group of cat- and dog-allergic patients, 73% had also specific IgE to guinea-pig dander. However, only 27% of the cat /dog-allergic patients had specific IgE to any of the non-cross-reactive guinea-pig allergens Cav p 1, Cav p 2 or Cav p 3. The high prevalence of IgE to guinea-pig dander could be explained by IgE-cross-reactivity among serum albumins and certain lipocalins., Conclusions and Clinical Relevance: The availability of specific allergen markers is essential for the assessment of primary sensitization, especially in polysensitized patients. The proposed panel of guinea-pig allergens Cav p 1, Cav p 2 and Cav p 3 is a first step to component-resolved IgE-diagnosis of allergy to small furry pets., (© 2021 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Haemaphysalis longicornis is distributed worldwide and transmits a variety of pathogens, causing human and animal disease. Use of chemical acaricides, as a primary tick control method, has several disadvantages, including acaricide resistance, environmental damage and residue accumulation in livestock. Development of a livestock vaccination aimed at a tick protective antigen could be an effective, labor-saving and environmentally-friendly method of reducing tick infestation and transmission of tick-borne pathogens. Lipocalins are low molecular weight proteins that play important roles in blood feeding, immune response and reproduction in ticks. In our study, the open reading frame (ORF) of a lipocalin homologue from H. longicornis (HlLIP) was successfully cloned, which consisted of 387 bp encoding a protein of 128 amino acids. The HlLIP protein sequence showed a close sequence homology with Ixodes persulcatus lipocalin. The HlLIP gene was constitutively detected in all developmental stages and in all tissues of the unfed female tick. The ORF of the HlLIP gene was sub-cloned into pET-32a (+) to obtain the recombinant protein (rHlLIP) and its immunogenicity was comfirmed by western blot. A vaccination trial on rabbits against H. longicornis infestation demonstrated that the rHlLIP protein could significantly prolong the period of tick blood feeding, and reduce tick engorged weight, oviposition and egg hatching rate. The vaccination efficacy of the rHlLIP protein was 60.17 % based on engorged weight, oviposition and egg hatching rate of ticks. The results obtained in this study demonstrate that rHlLIP protein is a promising antigen that could potentially be developed as a vaccine against H. longicornis infestation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Patients are commonly challenged with foods containing baked milk, for example muffins, yet little is known about the specific allergen content of muffins used in milk challenges or of the effect that baking has on allergenicity., Objective: Our objective was to compare the levels of major milk allergens in uncooked and baked muffins using monoclonal immunoassays and IgE antibody binding before and after baking., Methods: Uncooked and baked muffins were prepared using recipes from Mount Sinai and Imperial College. Allergen levels were compared by ELISA for Bos d 5 (β-lactoglobulin) and Bos d 11 (β-casein). IgE reactivity was assessed using sera from milk-sensitized donors in direct binding and inhibition ELISA., Results: Bos d 5 was reduced from 680 µg/g in uncooked muffin mix to 0.17 µg/g in baked muffins, representing a >99% decrease after baking. Conversely, Bos d 11 levels in baked muffin remained high and only decreased by 30% from a mean of 4249 µg/g in uncooked muffin mix to 2961 µg/g when baked (~181 mg Bos d 11 per muffin). Baked muffins retained ~70% of the IgE binding to uncooked muffin mix. Baked muffin extract inhibited IgE binding to uncooked muffin mix by up to 80%, demonstrating retention of in vitro IgE reactivity., Conclusions and Clinical Relevance: High levels of Bos d 11 in baked muffins pose a risk for adverse reactions, especially in patients who have high anti-casein IgE antibodies., (© 2020 John Wiley & Sons Ltd.)

Although some progress has been achieved in understanding certain aspects of the allergenic mechanism of animal lipocalins, they still remain largely enigmatic. One possibility to unravel this property is to investigate their interaction with components of the immune system. Since these components are highly complex we intended to use a high-throughput technology for this purpose. Therefore, we used phage-display of a random peptide library for panning against the dog allergen Can f 1. By this method we identified a Can f 1 binding peptide corresponding to the antigen-binding site of a putative γδT-cell receptor. Additional biochemical investigations confirmed this interaction., (© 2020 Matthias Habeler and Bernhard Redl, published by De Gruyter, Berlin/Boston.)

Human lipocalin 6 (hLCN6) is a newly discovered epididymal-specific secreted protein, capable of binding to the head and tail of spermatozoa and involved in sperm maturation. Anti-hLCN6 monoclonal antibody coupled immunomagnetic beads (IMBs) can be effectively used for the separation and forensic identification of sperm cells from mixed stains. But the source of monoclonal antibody is limited. In this study, an immunized mouse phage display antibody library was constructed and the single-chain variable fragments (scFvs) against hLCN6 were screened. The selection was performed using four rounds of biopanning and positive clones were validated by phage ELISA. Two anti-hLCN6 scFv clones with highest affinity were selected and sequencing result showed that the two sequences were identical. After prokaryotic expression and purification, the purified scFv could specifically recognize the hLCN6 in the lysate of human sperm cells and epididymis by western blot analysis, without any cross-reactivity with cellular antigens in female epithelial cells. The dissociation constant (K d ) of anti-hLCN6 scFv was 6.69 × 10 -7  mol/L measured by indirect ELISA. Therefore, our work not only provides a useful tool for further exploration of the biological functions of hLCN6, but also opens up new research avenues for the separation of sperm cells from mixed stains based on immuno-binding reaction., Competing Interests: Declaration of competing interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Molecular ON-switches in which a chemical compound induces protein-protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics., Competing Interests: Competing interest statement: The authors declare competing financial interests. C.U.Z., C.O., M.W.T, and M.L. have filed a patent application related to the technology described in this article. The value of this patent application may be affected by publication of this article. The other authors declare no competing interests., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: How T H 2-mediated allergic immune responses are induced is still under investigation., Objective: In an in vitro system we compared the effect of lipocalin allergens and nonallergenic homologues on human monocyte-derived dendritic cells (DCs) to investigate how they polarize naive CD4 + T H cells. Microarray data gained with these DCs showed a significant difference in expression of formyl peptide receptors (FPRs). Activation of FPR3 in human monocyte-derived DCs leads to inhibition of IL-12 production. Low concentrations of IL-12 during T-cell priming biases immune responses toward T H 2. We hypothesize that binding of allergenic lipocalins to FPR3 might be a mechanism for induction of allergic immune responses., Methods: We examined whether lipocalins and FPR3 colocalize within the cells by using confocal microscopy. With calcium mobilization assays of FPR3-transfected HEK 293 cells, we measured FPR3 signaling in response to allergenic and nonallergenic lipocalins. Silencing of FPR3 in DCs and pretreatment with an antagonistic peptide were used to assess the function of FPR3 in T H 2 induction., Results: FPR3 and lipocalins colocalize in the same vesicles in DCs. Cathepsin S-digested allergenic lipocalins, but not digestion products of nonallergenic homologues, activate FPR3 signaling. FPR3 silencing in DCs or pretreatment with an antagonistic peptide restores IL-12 and induces IL-10 expression by DCs treated with lipocalin allergens, attenuating the T H 2 bias and inducing IL-10 production in cocultured T H cells., Conclusion: We describe a novel molecular mechanism for induction of T H 2-mediated allergic immune responses., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Frequently observed multiple sensitizations to several animals highlights the importance of a molecular diagnosis, distinguishing between sensitizations specific to single species and sensitizations due to cross-reactivity., Objective: The aim of our study was to assess the usefulness of a molecular diagnosis in the description of sensitization profiles in allergy patients living in Central Europe, with a particular focus on animal-derived molecules., Methods: The molecular diagnosis was performed using the ImmunoCAP ISAC microarray. Results of 1,255 allergy patients were subjected to statistical analysis., Results: The highest sensitization rates were observed for uteroglobin Fel d 1 (31.8%) and kallikrein Can f 5 (16.4%), followed by animal lipocalins Can f 1 (13.9%), Equ c 1 (6.2%), Fel d 4 (5.3%), Can f 2 (4.2%), and Mus m 1 (4.1%). Sensitization rates to serum albumins Fel d 2, Can f 3, Equ c 3, and Bos d 6 were very low, with the highest being 3.2% to Fel d 2. Detailed subanalysis confirmed the dominant role of Fel d 1 or Can f 5 and/or Can f 1 in cat- or dog-sensitized patients, respectively. Further analysis focused on lipocalins and albumins confirmed a high rate of cosensitizations within both groups., Conclusion: The sensitization to animal allergen molecules is very frequent in Central Europe. The most common is sensitization to species-specific cat uteroglobin Fel d 1 and dog kallikrein Can f 5, followed by sensitizations to animal lipocalins. Our data suggest that commonly observed multiple sensitizations detected by extract approach can be explained not only by true cosensitization, but also by cross-reactivity, mainly in the frame of lipocalins. Cross-reactive serum albumins are minor sensitizers and are probably not important from this point of view., (© 2020 S. Karger AG, Basel.)

The use of recombinant allergens is a promising approach in allergen-specific immunotherapy (AIT). Considerable limitation, however, has been the ability of recombinant allergens to activate effector cells leading to allergic reactions. Recombinant hypoallergens with preserved protein folding and capacity to induce protective IgG antibodies binding effectively to the native allergen upon sensitization would be beneficial for safer AIT. In this study, hypoallergen variants of the major horse allergen Equ c 1 were designed by introducing one point mutation on the putative IgE epitope region and two mutations on the monomer-monomer interface of Equ c 1 dimer. The recombinant Equ c 1 wild type and the variants were produced and purified to homogeneity, characterized by size-exclusion ultra-high performance liquid chromatography and ultra-high resolution mass spectrometry. The IgE-binding profiles were analyzed by a competitive immunoassay and the biological activity by a histamine release assay using sera from horse allergic individuals. Two Equ c 1 variants, Triple 2 (V47K + V110E + F112K) and Triple 3 (E21Y + V110E + F112K) showed lower allergen-specific IgE-binding capacity and decreased capability to release histamine from basophils in vitro when using sera from six allergic individuals. Triple 3 showed higher reduction than Triple 2 in IgE-binding (5.5 fold) and in histamine release (15.7 fold) compared to wild type Equ c 1. Mutations designed on the putative IgE epitope region and monomer-monomer interface of Equ c 1 resulted in decreased dimerization, a lower IgE-binding capacity and a reduced triggering of an allergic response in vitro.

Lipocalins represent the most important protein family of the mammalian respiratory allergens. Four of the seven named dog allergens are lipocalins: Can f 1, Can f 2, Can f 4, and Can f 6. We present the structure of Can f 6 along with data on the biophysical and biological activity of this protein in comparison with other animal lipocalins. The Can f 6 structure displays the classic lipocalin calyx-shaped ligand binding cavity within a central β-barrel similar to other lipocalins. Despite low sequence identity between the different dog lipocalin proteins, there is a high degree of structural similarity. On the other hand, Can f 6 has a similar primary sequence to cat, horse, mouse lipocalins as well as a structure that may underlie their cross reactivity. Interestingly, the entrance to the ligand binding pocket is capped by a His instead of the usually seen Tyr that may help select its natural ligand binding partner. Our highly pure recombinant Can f 6 is able to bind to human IgE (hIgE) demonstrating biological antigenicity., Competing Interests: The authors have declared that no competing interests exist.

Background: Horses are an important source of allergens, but the distribution of horse allergens is poorly understood. Five horse allergens have been identified, Equ c 1-4 and 6. Equ c 4 seems to be an important allergen, with an IgE-binding frequency of 77% in horse-sensitized individuals., Objectives: The aim of this study was to investigate levels of horse allergen Equ c 4 in dander, saliva and urine from ten horse breeds., Method: The study population included 170 horses (87 mares, 27 stallions, 56 geldings) from ten breeds. Horse dander, saliva and urine samples were collected. Levels of horse allergen Equ c 4 were quantified using a two-site sandwich ELISA (mAb 103 and 14G4) and were expressed as Equ c 4 U/μg protein., Results: The horse allergen Equ c 4 was present in all dander and saliva samples from ten horse breeds, with high within-breed and inter-breed variations; GM values were 639 Equ c 4 U/μg protein (range 5-15 264) for dander and 39.5 (4-263) for saliva. Equ c 4 was found in 19/21 urine samples. Adjusted for age, sex and changes over time, no differences between breeds could be seen in dander, while in saliva the North Swedish horse showed lower levels of Equ c 4 than any other breed. The levels of Equ c 4 protein in dander and saliva were significantly higher in samples from stallions compared to mares and geldings, independent of breed., Conclusions and Clinical Relevance: The results show a high variability in allergen levels of Equ c 4 in dander and saliva both within and between breeds. Significantly higher levels were found in stallions compared to mares and geldings, independent of breed. Results suggest that none of the horse breeds studied can be recommended for individuals allergic to Equ c 4., (© 2019 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET‑28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS‑PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B‑cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog‑allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0‑fold increase in cluster of differentiation 63 and C‑C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog‑allergic children compared with those of non‑allergic controls. The secondary structure analysis showed that Can f 7 contains 6 β‑sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.

Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.

Stable mutualism between a host and its resident bacteria requires a moderated immune response to control bacterial population size without eliciting excessive inflammation that could harm both partners. Little is known about the specific molecular mechanisms utilized by bacterial mutualists to temper their hosts' responses and protect themselves from aggressive immune attack. Using a gnotobiotic larval zebrafish model, we identified an Aeromonas secreted immunomodulatory protein, AimA. AimA is required during colonization to prevent intestinal inflammation that simultaneously compromises both bacterial and host survival. Administration of exogenous AimA prevents excessive intestinal neutrophil accumulation and protects against septic shock in models of both bacterially and chemically induced intestinal inflammation. We determined the molecular structure of AimA, which revealed two related calycin-like domains with structural similarity to the mammalian immune modulatory protein, lipocalin-2. As a secreted bacterial protein required by both partners for optimal fitness, AimA is an exemplar bacterial mutualism factor., Competing Interests: AR Have two patent applications related to AimA pending in the United States, Europe, and Canada (US Patent App. 15/883,999 and 15/311,667). ES, LK, MD, AP, AB, MH No competing interests declared, KG Karen Guillemin: Have two patent applications related to AimA pending in the United States, Europe, and Canada (US Patent App. 15/883,999 and 15/311,667)., (© 2018, Rolig et al.)

Background: Sensitization to dog dander is an important risk factor for rhinoconjunctivitis and asthma but is not sufficient for diagnosing dog allergy. Molecular allergy diagnostics offer new opportunities for refined characterization., Objectives: We sought to study the association between sensitization to all presently known dog allergen components and clinical symptoms of dog allergy in children evaluated by using nasal provocation tests (NPTs)., Methods: Sixty children (age, 10-18 years) sensitized to dog dander extract underwent NPTs with dog dander extract. Measurement of IgE levels to dog dander and to Can f 1, Can f 2, Can f 3, and Can f 5 was performed with ImmunoCAP, and measurement of IgE levels to Can f 4 and Can f 6 was performed with streptavidin ImmunoCAP. An IgE level of 0.1 kU A /L or greater was considered positive., Results: There was an association between sensitization to an increasing number of dog allergen components and a positive nasal challenge result (P = .01). Sensitization to lipocalins (odds ratio [OR], 6.0; 95% CI, 1.04-34.5), in particular Can f 4 (OR, 6.80; 95% CI 1.84-25.2) and Can f 6 (OR, 5.69; 95% CI, 1.59-20.8), was associated with a positive NPT result. Monosensitization to Can f 5 was related to a negative NPT result (OR, 5.78; 95% CI, 1.01-33.0)., Conclusion: Sensitization to an increasing number of dog allergen components and to lipocalins is associated with dog allergy. Monosensitization to Can f 5 should not be regarded primarily as a marker for dog allergy., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Increasing dairy consumption in China has been accompanied by rising incidence of milk allergy. Here we analyzed profiles of specific immunoglobulin E (sIgE) against cow's milk proteins, and assessed their value for milk allergy diagnosis among infants and young children from northern China., Methods: Sera collected from 48 patients with milk allergy and 27 negative control subjects was analyzed by enzyme-linked immunosorbent assay to measure sIgE to α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5), α-casein (Bos d 9), β-casein (Bos d 11), and κ-casein (Bos d 12)., Results: Among milk-allergic individuals, most were sensitized to at least one milk protein; about half were sensitized to Bos d 5, Bos d 9, Bos d 11 and Bos d 12, respectively, while few had positive serum sIgE against Bos d 4. Bos d 12 sIgE had the largest area under curve (AUC) (0.878; 95% CI, 0.800-0.957) and thus showed the best diagnostic performance in discriminating between milk-allergic and non-milk allergic patients, with a sensitivity of 92.6% and specificity of 72.9% using a statistically optimal cut-off value (OD 450nm , 0.191). The combinations of Bos d 5 + Bos d 12 showed an AUC of 0.926, which was larger than for any individual components., Conclusions: Our results revealed inter-individual variation in the sensitization to different milk allergen component. Bos d 12 sIgE showed best performance in diagnosing milk allergy. Milk allergy diagnostic accuracy was further improved using combinations of milk allergen components by application of ROC curves based on logistic regression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The major cow's milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of -7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Human lipocalin 6 (hLCN6) is a member of the lipocalin family, which is a group of structurally conserved hydrophobic ligand binding proteins, and widely distributed in animal, plant, and bacteria. Specific expression of hLCN6 in the epididymis and localization of this protein on the surface of spermatozoa suggest a role played by hLCN6, which may function as a transporter to carry ligands in the epididymal channel. However, the role of hLCN6 in sperm maturation has been largely unknown due to the lack of effective antibodies. In this study, we report the prokaryotic expression, purification, and refolding of recombinant hLCN6. Purified hLCN6 protein was used to generate monoclonal antibody (mAb) against this protein using conventional hybridoma techniques. The sensitivity and specificity of the anti-hLCN6 mAb were determined based on their activities in enzyme-linked immunosorbent assay and Western blotting analysis using various human tissues. The results showed that the antibody induced by recombinant hLCN6 protein had high sensitivity and specificity. Taken together, the recombinant hLCN6 protein and mAb against this protein obtained from our study provided useful tools for further exploration of the biological functions and molecular mechanism, as well as pathological significance of LCN6 in human.

Quinolinic acid, a macrophage/microglia-derived excitotoxin fulfills a plethora of functions such as neurotoxin, gliotoxin, and proinflammatory mediator, and it alters the integrity and cohesion of the blood-brain barrier in several pathophysiological states. Beta-trace protein (BTP), a monomeric glycoprotein, is known to indicate cerebrospinal fluid leakage. Thus, the prior aim of this study was to investigate whether BTP might non-invasively indicate quinolinic acid-induced impaired blood-brain barrier integrity. The research hypotheses were tested in three subsamples with different states of immune activation (patients with HCV-infection and interferon-α, patients with major depression, and healthy controls). BTP has also been described as a sensitive marker in detecting impaired renal function. Thus, the renal function has been considered. Our study results revealed highest quinolinic acid and highest BTP- levels in the subsample of patients with HCV in comparison with the other subsamples with lower or no immune activation (quinolinic acid: F = 21.027, p < 0.001 [ANOVA]; BTP: F = 6.792, p < 0.01 [ANOVA]). In addition, a two-step hierarchical linear regression model showed that significant predictors of BTP levels are quinolinic acid, glomerular filtration rate and age. The neurotoxin quinolinic acid may impair blood-brain barrier integrity. BTP might be a new non-invasive biomarker to indicate quinolinic acid-induced impaired blood-brain barrier integrity.

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat-sensitized Swedish patients and elucidated its allergenic activity and cross-reactivity with the dog lipocalin Can f 1. Sixty-five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross-reactive antisera. Fel d 7 is a common allergen in a Swedish cat-sensitized population that cross-reacts with Can f 1, and may contribute to symptoms in cat- but also in dog-allergic patients., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Unlabelled: The distinction between acute infections of bacterial or viral causes is clinically important, but often very difficult even for experienced doctors. Previous studies indicated that serum measurements of HNL (Human Neutrophil Lipocalin) might be a superior diagnostic means in this regard, but also indicated that the antibody conformation of the HNL assay might have an impact on the diagnostic performance. The aim of the present report was to examine this further., Methods: Several different (n=24) HNL ELISA assays were developed using different combinations of monoclonal and polyclonal HNL antibodies. Sera were collected from healthy persons (n=188) and from 155 patients with acute infections before any antibiotics treatment. The patients were diagnosed as having bacterial (n=69) or viral causes (n=86) of their infections. Plasma and serum were also examined by Western blotting using HNL-specific polyclonal antibodies., Results: The optimal assay format for the distinction between bacterial and viral infection resulted in an area under the receiver operating characteristics curve (AuROC) for S-HNL of 0.98. (95% CI 0.94-1.00) as compared to 0.83 (0.76-0.88) for blood neutrophil counts and 0.69 (0.61-0.76) for S-CRP. Results also showed that different assay formats of HNL identified monomeric and dimeric HNL differently, the monomeric HNL being elevated in viral infections and the dimeric HNL being elevated in bacterial infections., Conclusion: We conclude that serum measurement of HNL is a superior diagnostic means to distinguish between acute infections caused by bacteria or virus. For optimal clinical performance the immunoassay should address conformational epitopes in the dimeric HNL., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the γ-emitter (123) I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging., (© 2015 UICC.)

Owners and their domestic animals via skin shedding and secretions, mutually exchange microbiomes, potential pathogens and innate immune molecules. Among the latter especially lipocalins are multifaceted: they may have an immunomodulatory function and, furthermore, they represent one of the most important animal allergen families. The amino acid identities, as well as their structures by superposition modeling were compared among human lipocalins, hLCN1 and hLCN2, and most important animal lipocalin allergens, such as Can f 1, Can f 2 and Can f 4 from dog, Fel d 4 from cats, Bos d 5 from cow's milk, Equ c 1 from horses, and Mus m 1 from mice, all of them representing major allergens. The β-barrel fold with a central molecular pocket is similar among human and animal lipocalins. Thereby, lipocalins are able to transport a variety of biological ligands in their highly conserved calyx-like cavity, among them siderophores with the strongest known capability to complex iron (Fe(3+) ). Levels of human lipocalins are elevated in nonallergic inflammation and cancer, associated with innate immunoregulatory functions that critically depend on ligand load. Accordingly, deficient loading of lipocalin allergens establishes their capacity to induce Th2 hypersensitivity. Our similarity analysis of human and mammalian lipocalins highlights their function in innate immunity and allergy., (© 2015 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Problem: A wide variety of mediators are involved in inflammatory processes. However, the identity of those participating in vaginal immune responses has not been established. We correlated extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase-8 (MMP-8), hyaluronan (HA), hyaluronidase-1 (Hyal-1), human β-defensin-2 (hBD2), and neutrophil gelatinase-associated lipocalin (NGAL) concentrations with the extent of leukocyte infiltration into the vagina and suggest their participation in vaginal inflammation., Methods of Study: Vaginal fluid was obtained from 233 women seen at the outpatient clinic in the Department of Obstetrics and Gynecology at Campinas University, Brazil. The magnitude of vaginal inflammation was determined by the leukocyte count on vaginal smears and categorized as no inflammation (0 leukocytes/field), moderate inflammation (1-4 leukocytes/field), and intense inflammation (>4 leukocytes/field). Concentrations of EMMPRIN, MMP-8, HA, Hyal-1, hBD2, and NGAL were determined in vaginal fluid by ELISA., Results: EMMPRIN, MMP-8, HA, hBD2, and NGAL concentration increased with elevated leukocyte numbers (P < 0.05), while Hyal-1 did not. EMMPRIN concentrations were correlated with HA and MMP-8 levels., Conclusion: EMMPRIN, MMP-8, HA, β-defensin, and NGAL are elevated in women with vaginal inflammation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Lipocalins seem to explain the cross-reactivity between some pets such as cat and dog. However, its role in other animals and its possible clinical impact in allergy diseases have been scarcely studied., Objective: To analyze by bioinformatics techniques, the identity between lipocalin of some animals and to explore the clinical impact on allergic diseases., Material and Method: An in silico study was done to search for lipocalin sequences using the BLAST program of NCBI Database. The protein sequences were aligned with CLUSTAL Omega UniProt version 1.2.1 software. The base sequences for alignments were lipocalins dogs and cats. The defined percentage identity was compared with the frequency of sensitization to animals exposed in a population of 284 patients with suspected allergic diseases., Results: Identities between sequences were 10% to 70%. The highest values were found with Can f 6-Fel d 4 (68%) and Fel d 4-Equ c 1 (68%). The lower identity was found with lipocalin porpurin and retinol binding (<20%). We observed a relationship between sensitization and the percent identity between the species studied., Conclusion: Lipocalins as Can f 6, Fel de 4 and Equ c 1 seem to play an important role in the cross-reactivity to cat, horse and dog but not for the co-sensitization to hamster, cow or birds. Fel de 4 and Equ c 1 could be a prevalent allergen for horse and cat. These results come from predictive analysis and must be confirmed by in vitro and in vivo studies.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The recently identified dog lipocalin allergen Can f 4 is an important respiratory allergen., Objective: We sought to comprehensively characterize the memory CD4(+) T-cell responses of allergic and nonallergic subjects to Can f 4., Methods: Can f 4-specific CD4(+)CD45RO(+) T-cell lines (TCLs) from allergic and healthy subjects were established and characterized by their functional and phenotypic properties. The epitope specificity of the TCLs was tested with 48 overlapping 16-mer peptides spanning the sequence of Can f 4. HLA restriction of the specific TCLs and the binding capacity of the epitope-containing peptides to common HLA class II molecules were studied., Results: Can f 4-specific memory CD4(+) TCLs were obtained at an 8-fold higher frequency from allergic than from nonallergic subjects. Functionally, the TCLs of allergic subjects exhibited a higher T-cell receptor avidity and expression of CD25 and predominantly produced IL-4 and IL-5. The TCLs of nonallergic subjects mostly secreted IFN-γ and IL-10, with high CXCR3 expression. Several distinct T-cell epitope regions along the allergen were identified. Importantly, the peptides from the region between amino acids 43 and 67 showed promiscuous HLA-binding capacity and induced memory CD4(+) T-cell responses in 90% of the allergic donors., Conclusion: Productive TH2-deviated memory T-cell responses to Can f 4 are observed in allergic but not nonallergic subjects. A 19-mer peptide sequence covering the core of the immunodominant region of the allergen is a potential target for the development of peptide-based allergen immunotherapy., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore, constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1's endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra(-/-) background. Conversely, IL-17-dependent pathology in Zc3h12a(+/-) mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3' UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ, and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Lipocalins are one of the most important groups of inhalant animal allergens. The analysis of structural features of these proteins is important to get insights into their allergenicity. We have determined two different dimeric crystal structures for bovine dander lipocalin Bos d 2, which was earlier described as a monomeric allergen. The crystal structure analysis of all other determined lipocalin allergens also revealed oligomeric structures which broadly utilize inherent structural features of the β-sheet in dimer formation. According to the moderate size of monomer-monomer interfaces, most of these dimers would be transient in solution. Native mass spectrometry was employed to characterize quantitatively transient dimerization of two lipocalin allergens, Bos d 2 and Bos d 5, in solution.

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Since bacterial invasion into host cells is a critical step in the infection process and the predominance of multiple-antibiotic-resistant Klebsiella (K.) pneumoniae strains, using molecular agents to interfere with K. pneumoniae invasion is an attractive approach for the prevention of infection and suppress the immune inflammatory response. In previous studies by our group, high-mobility group nucleosome-binding domain 2 (HMGN2) protein was shown to exhibit anti-bacterial activity in vitro. The objective of the present study was to investigate the effects of HMGN2 protein on the invasion of K. pneumoniae 03183 in vivo. The results showed that pre-treatment with 128 µg/ml HMGN2 significantly reduced K. pneumoniae 03183 invasion into mouse lungs and increased the mRNA expression of CXCL1 and LCN2 within 2 h. Immunohistochemical staining showed that F-actin expression was significantly decreased, and fluorescence microscopy and western blot analysis further demonstrated that HMGN2 significantly blocked K. pneumoniae 03183-induced actin polymerization. These changes implied that HMGN2 may provide protection against K. pneumoniae 03183 infection in vivo.

Background: Renal transplantation is the best choice for the treatment of dialysis patients with end-stage renal failure because it provides better quality of life and more life time. However, despite successful surgical techniques, immunological issues in kidney transplantation are not completely resolved. Thus, after transplantation, patients must be followed up closely. Although patient follow-up with the use of creatinine and renal biopsy are common, it is thought that biopsy is too invasive and that creatinine is unreliable. Hence, new parameters that correlate with the patient's immunological condition are needed in clinical monitoring., Methods: One of the biomarkers that has been studied recently is neutrophil gelatinase-associated lipocalin (NGAL). Its diagnostic value in cases of acute renal failure, delayed graft function, and IgA nephropathy is widely investigated. However, data are insufficient as to whether NGAL can be used for follow-up in the chronic process after renal transplantation. We aimed to investigate the predictive value of NGAL in terms of rejection in donor-specific antibody (DSA)-positive and DSA-negative renal transplant patients. Ninety patients were included., Results: We found that rejection rates were higher in patients whose NGAL values were ≥ 50 and DSA-positive. Delayed graft function was seen more frequently in patients whose NGAL values were ≥ 50., Conclusions: An increase in NGAL level does not always indicate renal injury because NGAL is also an acute-phase reactant. NGAL cannot be used alone to diagnose rejection, but, if NGAL level is high, it is necessary to study DSA, and sub-clinical rejection must be researched., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein., Methods: The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry)., Results: Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically., Conclusion: Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

Exposure of Anopheles gambiae mosquitoes to Plasmodium infection enhances the ability of their immune system to respond to subsequent infections. However, the molecular mechanism that allows the insect innate immune system to 'remember' a previous encounter with a pathogen has not been established. Challenged mosquitoes constitutively release a soluble haemocyte differentiation factor into their haemolymph that, when transferred into Naive mosquitoes, also induces priming. Here we show that this factor consists of a Lipoxin/Lipocalin complex. We demonstrate that innate immune priming in mosquitoes involves a persistent increase in expression of Evokin (a lipid carrier of the lipocalin family), and in their ability to convert arachidonic acid to lipoxins, predominantly Lipoxin A4. Plasmodium ookinete midgut invasion triggers immune priming by inducing the release of a mosquito lipoxin/lipocalin complex.

Acute renal failure (ARF) represents a very important and potentially devastating disorder in clinical nephrology. Neutrophil gelatinase-associated lipocalin (NGAL) is an early biomarker for ARF in a wide range of different disease processes, which is frequently detected in clinical diagnosis. Herein, we present a label-free and sensitive photoelectrochemical (PEC) immunosensor for NGAL by utilizing a biotinylated anti-NGAL Nanobody (Nb) orientedly immobilized to streptavidin-coated cobalt 2,9,16,23-tetraaminophthalocyanine (CoPc)-sensitized TiO2 electrode. The Nb was biotinylated at the C-terminus, which is situated at the opposite site of the antigen binding region. Using highly oriented Nb as receptor molecules, a label-free PEC immunosensor for NGAL was developed by monitoring the changes in the photocurrent signals of the electrode resulting from immunoreaction. Immobilization of Nb to streptavidin-coated CoPc-sensitized TiO2 electrode surface provides high binding capacity to NGAL; thus, it can lead to a high sensitivity. The limit of detection (LOD) of the proposed immunosensor has been significantly lowered to 0.6 pg mL(-1). This proposed immunosensor reveals high specificity to detect NGAL, with acceptable intra-assay precision and excellent stability. In addition, the present work provides a new approach to design Nb-based PEC immunosensor and increases versatility of Nbs.

Coxsackievirus strain B serotype 3 (CVB3)-induced myocarditis is an important human disease that causes permanent tissue damage and can lead to death from acute infection or long-term morbidity caused by chronic inflammation. The timing and magnitude of immune activation following CVB3 infection can mediate a positive host outcome or increase tissue pathology. To better elucidate the role of endosomal Toll-like receptor (TLR) signaling in acute CVB3 infection, we studied mice with a loss-of-function mutation, known as Letr for 'loss of endosomal TLR response', in Unc93b1, which is a chaperone protein for TLR3, TLR7 and TLR9. Using Unc93b1(Letr/)(Letr) mice, we determined that Unc93b1-dependent TLR activation was essential for the survival of acute CVB3-induced myocarditis. We also determined that a lack of endosomal TLR signaling was associated with a higher viral load in target organs and that it increased inflammation, necrosis and fibrosis in cardiac tissue. Loss of Unc93b1 function was also associated with increased cardiac expression of Ifn-b and markers of tissue injury and fibrosis including Lcn2 and Serpina3n early after CVB3 infection. These observations establish a significant role for Unc93b1 in the regulation of the host inflammatory response to CVB3 infection and also reveal potential mediators of host tissue damage that merit further investigation in acute viral myocarditis., (© 2015 S. Karger AG, Basel.)

Lipocalin-2 (LCN2), a secretory protein, regulates diverse cellular processes such as cell death/survival, cell migration/invasion, cell differentiation, iron delivery, inflammation, insulin resistance, and tissue regeneration. Recently, we reported that LCN2 is secreted by brain astrocytes under inflammatory conditions and that it promotes apoptosis, morphological changes, and migration in astrocytes both in vitro and in vivo. Activated astrocytes release LCN2 not only to induce the morphological transformation associated with reactive astrocytosis, but also to promote their own death. Under inflammatory conditions, activated astrocytes also show functional dichotomy similar to the M1/M2 phenotypes of microglia and macrophages. LCN2 is thought to be a chemokine inducer and an autocrine promoter of the classical proinflammatory activation of astrocytes. This article summarizes the current knowledge regarding the role of astrocyte-derived LCN2 as a proinflammatory mediator in the central nervous system and discusses LCN2’s role in neuroinflammatory disorders.

Background: Lipocalin 2 (LCN2), a protein primarily produced by hepatocytes, is highly upregulated under various conditions that induce cellular stress, such as intoxication, infection or inflammation. However, the precise biological functions and underlying mechanisms of LCN2 in hepatocytes remains unknown., Methods: Hepatocyte stress was successfully induced by treating Huh7 cells with interleukin-1β (IL-1β). Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α) and LCN2 levels were measured in IL-1β treated Huh7 cells and supernatant. Additionally, microarray analysis was conducted to identify genes differentially expressed in LCN2-silenced and control Huh7 cells., Results: TNF-α, IL-6 and LCN2 were significantly elevated in Huh7 cells after IL-1β) treatment. In LCN2-silenced Huh7 cells, expression of IL-6 and TNF-α was significantly increased when compared with the expression levels of control Huh7 cells. Furthermore, differentially expressed genes were observed between the LCN2-silenced and control cells. Microarray analysis indicated that LCN2 acted by influencing genes involved in protein metabolism, stress response, cell cycle and proliferation., Conclusions: Our results suggest that LCN2 upregulation protects hepatocytes from IL-1β-induced stress. Additionally, our microarray analysis of LCN2-silenced and control cells provides a better understanding of the mechanisms that may be influenced by LCN2 induction., (© 2015 S. Karger AG, Basel.)

Background: An increasing number of asthma cases upon exposure to hamsters and anaphylactic reactions following hamster bites are being reported, but the allergens responsible are still poorly characterized. In the Golden hamster, male-specific submaxillary gland protein (MSP), a lipocalin expressed in a sex- and tissue-specific manner in the submaxillary and lacrimal glands, is secreted in the saliva, tears and urine. The purpose of this study was to determine if MSP is an allergen, to identify IgE-reactive proteins of different hamster species and to analyse potential cross-reactivities., Methods: Fur extracts were prepared from four hamster species. Hamster-allergic patients were selected based on a history of positive IgE-test to hamster epithelium. The IgE-reactivity of patients' sera was investigated by means of immunoblot and ELISA. IgE-reactive proteins in fur extracts and the submaxillary gland were identified using anti-MSP antibodies, Edman sequencing or mass spectrometry. MSP was purified from Golden hamster and recombinant MSP was expressed in E. coli., Results: Four patients had IgE-antibodies against 20.5-kDa and 24-kDa proteins of Golden hamster fur extract, which were identified as MSP. IgE-reactive MSP-like proteins were detected in European hamster fur extract. Three patient sera showed IgE-reactive bands at 17-21 kDa in Siberian and Roborovski hamster fur extracts. These proteins were identified as two closely related lipocalins. Immunoblot inhibition experiments showed that they are cross-reactive and are different from MSP., Conclusion: MSP lipocalin of the Golden hamster was identified as an allergen, and it is different from the cross-reactive lipocalin allergens of Siberian and Roborovski hamsters. Our findings highlight the need for specific tools for the in vitro and in vivo diagnosis of allergy to different hamster species., (© 2015 S. Karger AG, Basel.)

Ischaemia-reperfusion (IR) injury is a major issue in cardiac transplantation. Inflammatory processes play a major role in myocardial IR injury. Lipocalin-2 (Lcn2), which is also known as neutrophil gelatinase-associated lipocalin, has multiple functions that include the regulation of cell death/survival, cell migration/invasion, cell differentiation and iron delivery. In our study, the hearts of C57BL/6 mice were flushed with and stored in cold Bretschneider solution for 8 h and then transplanted into a syngeneic recipient. We found that Lcn2 neutralization decreased the recruitment of neutrophils and macrophages. Troponin T (TnT) production, 24 h after myocardial IR injury, was reduced through anti-Lcn2 antibody administration. The cardiac output at 60 mmHg of afterload pressure was significantly increased in hearts administrated with anti-Lcn2 antibody administration (anti-Lcn-2: 58.9 ± 5.62 ml/min; control: 25.8 ± 4.1 ml/min; P < 0.05). Anti-Lcn2 antibody treatment suppressed M1 marker (IL-12, IL-23 and iNOS) expression but increased M2 marker (IL-10, Arg1 and Mrc1) expression. Furthermore, in our vitro and vivo experiments, we found that anti-Lcn2 antibody treatment failed to induce M1-related gene expression in response to LPS and that Lcn2 neutralization enhanced the expression of M2-related genes following IL-4 treatment. In conclusion, Lcn2 promotes M1 polarization, and Lcn2 neutralization attenuates cardiac IR injury., (© 2014 John Wiley & Sons Ltd.)

Background: Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality., Objective: To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog., Methods: A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production., Results: The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses., Conclusion: We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, β2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1., (© 2014 American Heart Association, Inc.)

Four out of six officially recognized dog allergens are members of the lipocalin protein family. So far, a three-dimensional structure has been determined for only one dog allergen, Can f 2, which is a lipocalin protein. We present here the crystal structure of a second lipocalin allergen from dog, a variant of Can f 4. Moreover, we have compared and analyzed the structures of these two weakly homologous (amino acid identity 21%) dog allergens. The size and the amino acid composition of the ligand-binding pocket indicate that Can f 4 is capable of binding only relatively small hydrophobic molecules which are different from those that Can f 2 is able to bind. The crystal structure of Can f 4 contained both monomeric and dimeric forms of the allergen, suggesting that Can f 4 is able to form transient (weak) dimers. The existence of transient dimers in solution was confirmed by use of native mass spectrometry. The dimeric structure of Can f 4 is formed when the ends of four β-strands are packed against the same strands from the second monomer. The residues in the interface are mainly hydrophobic and the formation of the dimer is similar to the major horse allergen Equ c 1. Interestingly, the crystal structure of dog Can f 2 has been reported to show a different type of dimer formation. The capability of these allergens to form dimers may be important for the development of immediate allergic reaction (mast cell activation) because oligomeric allergens can effectively present multivalent epitopes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The onset of autoantibodies in systemic autoimmunity can be the result of a breakdown in tolerance at multiple checkpoints. Genetic, hormonal, and immunological factors can combine with environmental influences to accelerate the onset of disease and aggravate disease outcome. Here, we describe a novel mechanism relating to the regulatory role of Neutrophil Gelatinase Associated Lipocalin (NGAL) in modulating the levels of autoantibodies in pristane induced lupus. Following a single injection of pristane intraperitoneally, NGAL expression was induced in both the serum and spleen. Furthermore, NGAL deficient mice were more susceptible to the induction of pristane stimulated autoimmunity, and displayed higher numbers of autoantibody secreting cells and increased expression of activation induced cytidine deaminase (AID) and other inflammatory mediators in the spleen. In contrast, kidney damage was milder in NGAL deficient mice, indicating that NGAL was detrimental in autoantibody mediated kidney disease. These studies indicate that NGAL plays differential roles in different tissues in the context of lupus, and suggest a previously unrecognized role for NGAL in adaptive immunity., (Copyright © 2014 Elsevier Inc. All rights reserved.)