Structure, Functions, and Implications of Selected Lipocalins in Human Disease.

The lipocalin proteins are a large family of small extracellular proteins that demonstrate significant heterogeneity in sequence similarity and have highly conserved crystal structures. They have a variety of functions, including acting as carrier proteins, transporting retinol, participating in olfaction, and synthesizing prostaglandins. Importantly, they also play a critical role in human diseases, including cancer. Additionally, they are involved in regulating cellular homeostasis and immune response and dispensing various compounds. This comprehensive review provides information on the lipocalin family, including their structure, functions, and implications in various diseases. It focuses on selective important human lipocalin proteins, such as lipocalin 2 (LCN2), retinol binding protein 4 (RBP4), prostaglandin D2 synthase (PTGDS), and α1-microglobulin (A1M).

EAACI Molecular Allergology User's Guide 2.0.

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

Role of Small Molecule Ligands in IgE-Mediated Allergy.

PURPOSE OF REVIEW: A significant fraction of allergens bind small molecular ligands, and many of these compounds are classified as lipids. However, in most cases, we do not know the role that is played by the ligands in the allergic sensitization or allergic effector phases. RECENT FINDINGS: More effort is dedicated toward identification of allergens' ligands. This resulted in identification of some lipidic compounds that can play active immunomodulatory roles or impact allergens' molecular and allergic properties. Four allergen families (lipocalins, NPC2, nsLTP, and PR-10) are among the best characterized in terms of their ligand-binding properties. Allergens from these four families are able to bind many chemically diverse molecules. These molecules can directly interact with human immune system and/or affect conformation and stability of allergens. While there is more data on the allergens and their small molecular ligands, we are just starting to understand their role in allergy.

Non-Immunoglobulin Synthetic Binding Proteins for Oncology.

Extensive application of technologies like phage display in screening peptide and protein combinatorial libraries has not only facilitated creation of new recombinant antibodies but has also significantly enriched repertoire of the protein binders that have polypeptide scaffolds without homology to immunoglobulins. These innovative synthetic binding protein (SBP) platforms have grown in number and now encompass monobodies/adnectins, DARPins, lipocalins/anticalins, and a variety of miniproteins such as affibodies and knottins, among others. They serve as versatile modules for developing complex affinity tools that hold promise in both diagnostic and therapeutic settings. An optimal scaffold typically has low molecular weight, minimal immunogenicity, and demonstrates resistance against various challenging conditions, including proteolysis - making it potentially suitable for peroral administration. Retaining functionality under reducing intracellular milieu is also advantageous. However, paramount to its functionality is the scaffold's ability to tolerate mutations across numerous positions, allowing for the formation of a sufficiently large target binding region. This is achieved through the library construction, screening, and subsequent expression in an appropriate system. Scaffolds that exhibit high thermodynamic stability are especially coveted by the developers of new SBPs. These are steadily making their way into clinical settings, notably as antagonists of oncoproteins in signaling pathways. This review surveys the diverse landscape of SBPs, placing particular emphasis on the inhibitors targeting the oncoprotein KRAS, and highlights groundbreaking opportunities for SBPs in oncology.

Apolipoprotein D as a Potential Biomarker in Neuropsychiatric Disorders.

Neuropsychiatric disorders (NDs) are a diverse group of pathologies, including schizophrenia or bipolar disorders, that directly affect the mental and physical health of those who suffer from them, with an incidence that is increasing worldwide. Most NDs result from a complex interaction of multiple genes and environmental factors such as stress or traumatic events, including the recent Coronavirus Disease (COVID-19) pandemic. In addition to diverse clinical presentations, these diseases are heterogeneous in their pathogenesis, brain regions affected, and clinical symptoms, making diagnosis difficult. Therefore, finding new biomarkers is essential for the detection, prognosis, response prediction, and development of new treatments for NDs. Among the most promising candidates is the apolipoprotein D (Apo D), a component of lipoproteins implicated in lipid metabolism. Evidence suggests an increase in Apo D expression in association with aging and in the presence of neuropathological processes. As a part of the cellular neuroprotective defense machinery against oxidative stress and inflammation, changes in Apo D levels have been demonstrated in neuropsychiatric conditions like schizophrenia (SZ) or bipolar disorders (BPD), not only in some brain areas but in corporal fluids, i.e., blood or serum of patients. What is not clear is whether variation in Apo D quantity could be used as an indicator to detect NDs and their progression. This review aims to provide an updated view of the clinical potential of Apo D as a possible biomarker for NDs.

Ferroptosis resistance determines high susceptibility of murine A/J strain to iron-induced renal carcinogenesis.

Cancer susceptibility is a critical factor in the understanding of carcinogenesis. Intraperitoneal (i.p.) injection of an iron chelate, ferric nitrilotriacetate (Fe-NTA), produces hydroxyl radicals via Fenton reaction to induce ferroptosis in renal proximal tubules. Rats or mice subjected to repeated i.p. injections of Fe-NTA develop renal cell carcinoma (RCC). To elucidate the molecular mechanisms that cause susceptibility to renal carcinogenesis, we first established an inter-strain difference in the susceptibility to Fe-NTA-induced renal carcinogenesis in mice. Based on a previous observation of a low incidence of RCC with this model in C57BL/6J strain mice, we investigated A/J strain mice here, which demonstrated significantly higher susceptibility to Fe-NTA-induced renal carcinogenesis. Homozygous deletion of the Cdkn2a/2b tumor suppressor locus was detected for the first time in A/J strain mice. Focusing on ferroptosis and iron metabolism, we explored the mechanisms involved that lead to the difference in RCC development. We compared the protective responses in the kidney of A/J and C57BL/6J strains after Fe-NTA treatment. After 3-week Fe-NTA treatment, A/J mice maintained higher levels of expression of glutathione peroxidase 4 and xCT (SLC7A11), leading to a lower level of lipid peroxidation. Simultaneously, A/J mice had decreased expression of transferrin receptor and increased expression of ferritin to greater degrees than C57BL/6 mice. After a single Fe-NTA injection, higher levels of oxidative cell damage and cytosolic catalytic Fe(II) were observed in C57BL/6J mice, accompanied by a greater increase in lipocalin-2. Lipocalin-2 deficiency significantly decreased oxidative renal damage. Our results suggest that a genetic trait favoring ferroptosis resistance contributes to high susceptibility to Fe-NTA-induced RCC in A/J strain.

Lysine crotonylation of DgTIL1 at K72 modulates cold tolerance by enhancing DgnsLTP stability in chrysanthemum.

Lysine crotonylation of proteins is a recently identified post-translational modification (PTM) in plants. However, the function of lysine-crotonylated proteins in response to abiotic stress in plants has not been reported. In this study, we identified a temperature-induced lipocalin-1-like gene (DgTIL1) from chrysanthemum and showed that it was notably induced in response to cold stress. Overexpression of DgTIL1 enhanced cold tolerance in transgenic chrysanthemum. Ubiquitin membrane yeast two-hybrid (MYTH) system and bimolecular fluorescence complementation (BIFC) assays showed that DgTIL1 interacts with a nonspecific lipid transfer protein (DgnsLTP), which can promote peroxidase (POD) gene expression and POD activity to reduce the accumulation of reactive oxygen species (ROS) and improve resistance to cold stress in DgnsLTP transgenic chrysanthemum. In addition, we found that DgTIL1 was lysine crotonylated at K72 in response to low temperature in chrysanthemum. Moreover, lysine crotonylation of DgTIL1 prevented DgnsLTP protein degradation in tobacco and chrysanthemum. Inhibition of DgnsLTP degradation by lysine crotonylation of DgTIL1 further enhanced POD expression and POD activity, reduced the accumulation of ROS under cold stress in DgTIL1 transgenic chrysanthemum, thus promoting the cold resistance of chrysanthemum.

Major urinary protein excreted in rodent hindpaw sweat.

Alternative roles for sweat production beyond thermoregulation, considered less frequently, include chemical signaling. We identified the presence of a well-established rodent urinary pheromone, major urinary protein (MUP) in sweat ductules of the footpad dermal skin of mice. A hindpaw sweat proteomic analysis in hindpaw sweat samples collected in rats and generated by unmyelinated axon activation, identified seven lipocalin family members including MUP and 19 additional unique proteins. Behavioural responses to sniffing male mouse foot protein lysates suggested avoidance in a subset of male mice, but were not definitive. Rodent hindpaw sweat glands secrete a repertoire of proteins that include MUPs known to have roles in olfactory communication.

Early recognition of cardiac surgery-associated acute kidney injury: lack of added value of TIMP2 IGFBP7 over short-term changes in creatinine (an observational pilot study).

BACKGROUND: For the detection of cardiac surgery-associated acute kidney injury (CS-AKI), the performance of urine tissue inhibitor of metalloproteinase 2 insulin-like growth factor-binding protein 7 (TIMP2 IGFBP7) has never been compared with that of very early changes in plasma creatinine (∆pCr). We hypothesized that, in the context of perioperative haemodilution, lack of postoperative decrease in pCr would be of honourable performance for the detection of CS-AKI. We therefore aimed at comparing these biomarkers and their kinetics (primary objective). As secondary objectives, we assessed plasma neutrophil gelatinase-associated lipocalin (pNGAL), cystatin C (pCysC) and urea (pUrea). We also determined the ability of these biomarkers to early discriminate persistent from transient CS-AKI. METHODS: Patients over 75 years-old undergoing aortic valve replacement with cardiopulmonary bypass (CPB) were included in this prospective observational study. Biomarkers were measured before/after CPB and at the sixth postoperative hour (H6). RESULTS: In 65 patients, CS-AKI occurred in 27 (42%). ∆pCr from post-CPB to H6 (∆pCrpostCPB-H6): outperformed TIMP2 IGFBP7 at H6 and its intra- or postoperative changes: area under the receiver operating characteristic curve (AUCROC) of 0.84 [95%CI:0.73-0.92] vs. ≤0.67 [95%CI:0.54-0.78], p ≤ 0.03. The AUCROC of pNGAL, pCysC and pUrea did not exceed 0.72 [95%CI:0.59-0.83]. Indexing biomarkers levels for blood or urine dilution did not improve their performance. Combining TIMP2 IGFBP7 and ∆pCrpostCPB-H6 was of no evident added value over considering ∆pCrpostCPB-H6 alone. For the early recognition of persistent CS-AKI, no biomarker outperformed ∆pCrpostCPB-H6 (AUCROC = 0.69 [95%CI:0.48-0.85]). CONCLUSIONS: In this hypothesis-generating study mostly testing early detection of mild CS-AKI, there was no evident added value of the tested modern biomarkers over early minimal postoperative changes in pCr: despite the common perioperative hemodilution in the setting of cardiac surgery, if pCr failed to decline within the 6 h after CPB, the development of CS-AKI was likely. Confirmatory studies with more severe forms of CS-AKI are required.

Phagomimetic action of antibiotics: Revisited. How do antibiotics know where to go?

Phagocytic cells know exactly where an infection is by following chemotactic signals. The phagocytosis of bacteria results in a 'respiratory burst' in which superoxide radicals are released. We have previously compared the release of reactive oxygen species (ROS) by antibiotics, during electron transfer reactions, to this event. Antibiotics in their normal bacterial environment, and ROS, are both increasingly implicated in purposeful signalling functions, rather than their more widely known roles in bacterial killing and molecular damage. Here, we extend our comparison between antibiotics and phagocytic cells to propose that antibiotics actively accumulate at a site of pathogen infection or tumour growth. A common link being virulent cellular growth. When this occurs, new proteins are secreted, aberrant iron acquisition takes place, and lipocalins are released. Each provide a mechanism by which antibiotics can bind, and be retained, at an active site of pathogen infection or tumour growth.

A Tetrahedral Boronic Acid Diester Formed by an Unnatural Amino Acid in the Ligand Pocket of an Engineered Lipocalin.

Boronic acids have long been known to form cyclic diesters with cis-diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p-borono-l-phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so-called Borocalin. Here we describe the X-ray analysis of its covalent complex with 4-nitrocatechol as a high-affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho-hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar-binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans.

Probe-dependence of competitive fluorescent ligand binding assays to odorant-binding proteins.

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers.

Urinary cell cycle arrest biomarkers and chitinase 3-like protein 1 (CHI3L1) to detect acute kidney injury in the critically ill: a post hoc laboratory analysis on the FINNAKI cohort.

BACKGROUND: Acute kidney injury (AKI) is a frequently occurring syndrome in critically ill patients and is associated with worse outcomes. Biomarkers allow early identification and therapy of AKI which may improve outcomes. Urine chitinase 3-like protein 1 (uCHI3L1) was recently identified as a promising urinary biomarker for AKI. In this multicenter study, we evaluated the diagnostic performance for AKI stage 2 or greater of uCHI3L1 in comparison with the urinary cell cycle arrest biomarkers urinary tissue inhibitor of metalloproteinases-2 (TIMP-2)•insulin-like growth factor-binding protein 7 (IGFBP7) measured by NephroCheck Risk®. METHODS: Post hoc laboratory study of the prospective observational FINNAKI study. Of this cohort, we included patients with stored admission urine samples and availability of serum creatinine at day 1 of admission. Patients who already had AKI stage 2 or 3 at ICU admission were excluded. AKI was defined and staged according to the KDIGO definition and staging system. The primary endpoint was AKI stage 2 or 3 at day 1. Biomarker performance was assessed by the area under the curve of the receiver operating characteristic curve (AUC). We assessed individual performance and different combinations of urine biomarkers. RESULTS: Of 660 included patients, 49 (7.4%) had AKI stages 2-3 at day 1. All urine biomarkers were increased at admission in AKI patients. All biomarkers and most combinations had AUCs < 0.700. The combination uCHI3L1•TIMP-2 was best with a fair AUC of 0.706 (0.670, 0.718). uCHI3L1 had a positive likelihood ratio (LR) of 2.25 which was comparable to that of the NephroCheck Risk® cutoff of 2.0, while the negative LR of 0.53 was comparable to that of the NephroCheck Risk® cutoff of 0.3. CONCLUSIONS: We found that uCHI3L1 and NephroCheck Risk® had a comparable diagnostic performance for diagnosis of AKI stage 2 or greater within a 24-h period in this multicenter FINNAKI cohort. In contrast to initial discovery and validation studies, the diagnostic performance was poor. Possible explanations for this observation are differences in patient populations, proportion of emergency admissions, proportion of functional AKI, rate of developing AKI, and observation periods for diagnosis of AKI.

Activation of six lipocalins genes' transcription under PCB18 stress in OsTIL-silenced Oryza sativa L.

The lipocalins genes have been assigned for involving in the responses of organisms to various stress factors. The function of lipocalins under PCB18 stress was addressed by pathway complementation in the Oryza sativa L. OsTIL-silenced mutant. The growth of wild type (WT) and OsTIL-silenced mutant (MT) callus were suppressed by PCB18, and MT varieties were inhibited more seriously than WT varieties. Meanwhile, only WT varieties showed "Hormesis" effect. Compared with WT (3 day > 90.0%, 6 day ≤45.5%), MT varieties kept high removing efficiency by HPLC analysis. Varied gene transcription after OsTIL silencing was demonstrated between two varieties, especially obvious under PCB stress. Silenced OsTIL induced more protective gene transcriptions by qPCR analysis, OsVDE at 3 day, OsCHL, OsZEP1, OsZEP2 and OsUN at 6 day and OsZEP2 at 9 day. PCB18 stress further irritated these genes transcription in MT varieties. The defense stagy in WT varieties was that the transcriptions of lipocalins were inhibited to reduce PCB18 accumulation and toxicity. OsTIL could effectively limit PCB18 accumulation and toxicity. After TIL lacking, OsCHL, OsZEP1, OsZEP2 and OsUN in mutant were strongly evoked to against PCB stress. Remarkably, OsUN and OsZEP2 gene expressions were responded to PCB18 stress in both two varieties.

Roles of NGAL and MMP-9 in the tumor microenvironment and sensitivity to targeted therapy.

Various, diverse molecules contribute to the tumor microenvironment and influence invasion and metastasis. In this review, the roles of neutrophil gelatinase-associated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) in the tumor microenvironment and sensitivity to therapy will be discussed. The lipocalin family of proteins has many important functions. For example when NGAL forms a complex with MMP-9 it increases its stability which is important in cancer metastasis. Small hydrophobic molecules are bound by NGAL which can alter their entry into and efflux from cells. Iron transport and storage are also influenced by NGAL activity. Regulation of iron levels is important for survival in the tumor microenvironment as well as metastasis. Innate immunity is also regulated by NGAL as it can have bacteriostatic properties. NGAL and MMP-9 expression may also affect the sensitivity of cancer cells to chemotherapy as well as targeted therapy. Thus NGAL and MMP-9 play important roles in key processes involved in metastasis as well as response to therapy. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

The role of urinary KIM-1, NGAL, CA19-9 and ß2-microglobulin in the assessment of ureteropelvic junction obstruction in adults.

PURPOSE: The objective of this study is to evaluate the diagnostic properties of urinary biomarkers in adults with ureteropelvic junction obstruction: KIM-1, NGAL, CA19-9, and ß2-microglobulin. We also assessed urinary biomarker concentrations following pyeloplasty. MATERIAL AND METHODS: We prospectively studied adults from December 2013 to February 2015. We included 47 patients with a mean age of 38.6 ± 12.7 years. Each patient provided four samples of voided urine for biomarker measurement, one at pre-operative consultation and the others at 1, 3, and 6 months of post-operative follow-up. The control group consisted of 40 healthy individuals with no hydronephrosis on ultrasound evaluation. RESULTS: KIM-1 had an area under the curve of 0.79 (95% CI 0.70-0.89), NGAL 0.71 (95% CI 0.61-0.83), CA19-9 0.70 (95% CI 0.60-0.81), and ß2-microgloblin 0.61 (95% CI 0.50-0.73). KIM-1 was the most sensitive marker with a cut-off of 170.4 pg/mg creatinine (sensitivity 91.4%, specificity 59.1%), whereas CA19-9 was the most specific with a cut-off of 51.3 U/mg creatinine (sensitivity 48.9%, specificity 88.0%). Urinary concentrations of biomarkers decreased after pyeloplasty. CONCLUSIONS: The evaluation of urinary biomarkers is useful in adults undergoing pyeloplasty. KIM-1, NGAL, and CA19-9 were elevated and significantly decreased after surgery.

HDL-associated ApoM is anti-apoptotic by delivering sphingosine 1-phosphate to S1P1 & S1P3 receptors on vascular endothelium.

BACKGROUND: High-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved. METHODS: Human umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed. RESULTS: The addition of HDL+ApoM or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL-ApoM had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex. CONCLUSIONS: Altogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.

A comparison of the effects of oral vs. intravenous hydration on subclinical acute kidney injury in living kidney donors: a protocol of a randomised controlled trial.

BACKGROUND: Optimal treatment for established renal failure is living donor kidney transplantation. However this pathway exposes healthy individuals to significant reduction in nephron mass via major surgical procedure. Laparoscopic donor nephrectomy is now the most common method for live donor transplantation, reducing both donor post-operative pain and recovery time. However this procedure exposes kidneys to additional haemodynamic stresses. It has been suggested that donor hydration-particularly the use of preoperative intravenous fluids-may counteract these stresses, reducing subclinical acute kidney injury and ultimately improving long-term renal function. This may be important in both preservation of donor renal function and recipient graft longevity. METHODS/DESIGN: A prospective single-centre single-blinded randomized controlled trial will be carried out to determine the effects of donor preoperative intravenous fluids. The primary outcome is donor subclinical acute kidney injury (defined as plasma NGAL, >153 ng/ml) on day 1 postoperatively. Secondary outcomes include intraoperative haemodynamics, recipient subclinical acute kidney injury, perioperative complications and donor sleep quality. Donors will be randomised into two groups: the intervention group will receive active pre-hydration consisting of three litres of intravenous Hartmann's solution between midnight and 8 am before morning kidney donation, while the control group will not receive this. Both groups will receive unlimited oral fluids until midnight, as is routine. Plasma NGAL will be measured at pre-specified perioperative time points, intraoperative haemodynamic data will be collected using non-invasive cardiac output monitoring and clinical notes will be used to obtain demographic and clinical data. The researcher will be blinded to the donor fluid hydration status. Blinded statistical analysis will be performed on an intention-to-treat basis. A prospective power calculation estimates a required sample size of 86 patients. DISCUSSION: This study will provide important data, as there is currently little evidence about the use of donor preoperative fluids in laparoscopic nephrectomy. It is hoped that the results obtained will guide future clinical practice. TRIAL REGISTRATION: This study has been approved by the West of Scotland Research Ethics Committee 3 (reference no. 14/WS/1160, 27 January 2015) and is registered with the International Standard Randomised Controlled Trial Number Register (reference no. ISRCTN10199225 , 20 April 2015).

EAACI Molecular Allergology User's Guide.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

Neuronal production of lipocalin-2 as a help-me signal for glial activation.

BACKGROUND AND PURPOSE: We explored the hypothesis that injured neurons release lipocalin-2 as a help me signal. METHODS: In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2, and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2-activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen-glucose deprivation and promote neuroplasticity. RESULTS: In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated glial fibrillary acid protein, brain-derived neurotropic factor, and thrombospondin-1. Conditioned media from lipocalin-2-treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2-treated microglia upregulated synaptophysin and post-synaptic density 95 (PSD95) and protected neurons against oxygen-glucose deprivation. CONCLUSIONS: These findings provide proof of concept that lipocalin-2 is released by injured neurons as a help me distress signal that activates microglia and astrocytes into potentially prorecovery phenotypes.