Treatment-related changes in serum neutrophil gelatinase-associated lipocalin (NGAL) in psoriatic arthritis: results from the PIPA cohort study.

OBJECTIVES: Obesity and psoriatic arthritis (PsA) have a complicated relationship. While weight alone does not cause PsA, it is suspected to cause worse symptoms. Neutrophil gelatinase-associated lipocalin (NGAL) is secreted through various cell types. Our objective was to assess the changes and trajectories in serum NGAL and clinical outcomes in patients with PsA during 12 months of anti-inflammatory treatment. METHOD: This exploratory prospective cohort study enrolled PsA patients initiating conventional synthetic or biological disease-modifying anti-rheumatic drugs (csDMARDs/bDMARDs). Clinical, biomarker, and patient-reported outcome measures were retrieved at baseline, and 4 and 12 months. Control groups at baseline were psoriasis (PsO) patients and apparently healthy controls. The serum NGAL concentration was quantified by a high-performance singleplex immunoassay. RESULTS: In total, 117 PsA patients started a csDMARD or bDMARD, and were compared indirectly at baseline with a cross-sectional sample of 20 PsO patients and 20 healthy controls. The trajectory in NGAL related to anti-inflammatory treatment for all included PsA patients showed an overall change of -11% from baseline to 12 months. Trajectories in NGAL for patients with PsA, divided into treatment groups, showed no clear trend in clinically significant decrease or increase following anti-inflammatory treatment. NGAL concentrations in the PsA group at baseline corresponded to the levels in the control groups. No correlation was found between changes in NGAL and changes in PsA outcomes. CONCLUSION: Based on these results, serum NGAL does not add any value as a biomarker in patients with peripheral PsA, either for disease activity or for monitoring.

Neutrophil Gelatinase-Associated Lipocalin in Peritoneal Dialysis-Related Peritonitis: Correlation with White Blood Cells over Time and a Possible Role as the Outcome Predictor.

INTRODUCTION: The present study aimed to monitor peritoneal neutrophil gelatinase-associated lipocalin (pNGAL) during peritonitis episodes and to enhance its diagnostic value by evaluating pNGAL at scheduled times in parallel with white blood cell (WBC) count. In addition, we investigated possible correlations between pNGAL and the etiology of peritonitis, evaluating it as a possible marker of the clinical outcome. METHODS: Twenty-two patients with peritoneal dialysis (PD)-related peritonitis were enrolled. Peritonitis was divided into Gram-positive, Gram-negative, polymicrobial, and sterile. WBC count and neutrophil gelatinase-associated lipocalin (NGAL) in PD effluent were measured at different times (days 0, 1, 5, 10, 15, and/or 20 and 10 days after antibiotic therapy discontinuation). NGAL was measured by standard quantitative laboratory-based immunoassay and by colorimetric NGAL dipstick (NGALds) (dipstick test). RESULTS: We found strong correlations between peritoneal WBC, laboratory-based NGAL, and NGALds values, both overall and separated at each time point. On day 1, we observed no significant difference in WBC, both NGALds (p = 0.3, 0.9, and 0.2) between Gram-positive, Gram-negative, polymicrobial, and sterile peritonitis. No significant difference has been found between de novo versus relapsing peritonitis for all markers (p > 0.05). We observed a parallel decrease of WBC and both NGAL in patients with favorable outcomes. WBC count and both pNGAL resulted higher in patients with negative outcomes (defined as relapsing peritonitis, peritonitis-associated catheter removal, peritonitis-associated hemodialysis transfer, peritonitis-associated death) at day 10 (p = 0.04, p = 0.03, and p = 0.05, respectively) and day 15 (p = 0.01, p = 0.04, and tendency for p = 0.005). There was a tendency toward higher levels of WBC and NGAL in patients with a negative outcome at day 5. No significant difference in all parameters was proven at day 1 (p = 0.3, p = 0.9, p = 0.2) between groups. CONCLUSION: This study confirms pNGAL as a valid and reliable biomarker for the diagnosis of PD-peritonitis and its monitoring. Its trend is parallel to WBC count during peritonitis episodes, in particular, patients with unfavorable outcomes.

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.

Sepsis-mediated renal dysfunction: Pathophysiology, biomarkers and role of phytoconstituents in its management.

Sepsis has evolved as an enormous health issue amongst critically ill patients. It is a major risk factor that results in multiple organ failure and shock. Acute kidney injury (AKI) is one of the most frequent complications underlying sepsis, which portends a heavy burden of mortality and morbidity. Thus, the present review is aimed to provide an insight into the recent progression in the molecular mechanisms targeting dysregulated immune response and cellular dysfunction involved in the development of sepsis-associated AKI, accentuating the phytoconstituents as eligible candidates for attenuating the onset and progression of sepsis-associated AKI. The pathogenesis of sepsis-mediated AKI entails a complicated mechanism and is likely to involve a distinct constellation of hemodynamic, inflammatory, and immune mechanisms. Novel biomarkers like neutrophil gelatinase-associated lipocalin, soluble triggering receptor expressed on myeloid cells 1, procalcitonin, alpha-1-microglobulin, and presepsin can help in a more sensitive diagnosis of sepsis-associated AKI. Many bioactive compounds like curcumin, resveratrol, baicalin, quercetin, and polydatin are reported to play an important role in the prevention and management of sepsis-associated AKI by decreasing serum creatinine, blood urea nitrogen, cystatin C, lipid peroxidation, oxidative stress, IL-1ß, TNF-α, NF-κB, and increasing the activity of antioxidant enzymes and level of PPARγ. The plant bioactive compounds could be developed into a drug-developing candidate in managing sepsis-mediated acute kidney injury after detailed follow-up studies. Lastly, the gut-kidney axis may be a more promising therapeutic target against the onset of septic AKI, but a deeper understanding of the molecular pathways is still required.

Anticalin®-based therapeutics: Expanding new frontiers in drug development.

Anticalin proteins are a novel class of clinical-stage biopharmaceuticals with high potential in various disease areas. Anticalin proteins, derived from extracellular human lipocalins are single-chain proteins, with a highly stable structure that can be engineered to bind with high specificity and potency to targets of therapeutic relevance. The small size and stable structure support their development as inhalable biologics in the field of respiratory diseases as already demonstrated for PRS-060/AZD1402, an Anticalin protein currently undergoing clinical development for the treatment of asthma. Anticalin proteins provide formatting flexibility which allows fusion with the same or other Anticalin proteins, or with other biologics to generate multivalent, multiparatopic or multispecific fusion proteins. The fusion of Anticalin proteins to antibodies allows the generation of potent therapeutic proteins with new modes of action, such as antibody-Anticalin bispecific proteins with tumor-localized activity. Cinrebafusp alfa and PRS-344/S095012 antibody-Anticalin bispecific proteins were designed to reduce potential systemic toxicity by localizing the activity to the tumor, and are currently in clinical development in immuno-oncology. Furthermore, the ease in generating bi- and multispecifics as well as the small and stable structure prompted the investigation of Anticalin proteins for the CAR T space, opening additional potential treatment options based on Anticalin protein therapies.

Evaluation of renal injury in children with uncorrected CHDs with significant shunt using urinary neutrophil gelatinase-associated lipocalin.

BACKGROUND: CHDs can be complicated by renal injury which worsens morbidity and mortality. Urinary neutrophil gelatinase-associated lipocalin, a sensitive and specific biomarker of renal tubular injury, has not been studied in children with uncorrected CHDs. This study evaluated renal injury in children with uncorrected CHDs using this biomarker. METHODS: The patients were children with uncorrected CHDs with significant shunt confirmed on echocardiogram with normal renal ultrasound scan, in the paediatric cardiology clinic of a tertiary hospital. The controls were age-matched healthy children recruited from general practice clinics. Information on bio-data and socio-demographics were collected and urine was obtained for measurement of urinary neutrophil gelatinase-associated lipocalin levels. RESULTS: A total of 65 children with uncorrected CHDs aged 2 to 204 months were recruited. Thirty-one (47.7%) were males while 36 (55.4%) had acyanotic CHDs. The median urinary neutrophil gelatinase-associated lipocalin level of patients of 26.10 ng/ml was significantly higher than controls of 16.90 ng/ml (U = 1624.50, p = 0.023). The median urinary neutrophil gelatinase-associated lipocalin level of patients with cyanotic and acyanotic CHDs were 30.2 ng/ml and 22.60 ng/ml respectively; (Mann-Whitney U = 368.50, p = 0.116). The prevalence of renal injury using 95th percentile cut-off value of urinary neutrophil gelatinase-associated lipocalin was 16.9%. Median age of patients with renalinjury was 16 (4-44) months. CONCLUSIONS: Children with uncorrected CHDs have renal injury detected as early as infancy. The use of urinary neutrophil gelatinase-associated lipocalin in early detection of renal injury in these children may enhance early intervention and resultant prevention of morbidity and reduction in mortality.

Therapeutic Potential of Hematopoietic Prostaglandin D2 Synthase in Allergic Inflammation.

Worldwide, there is a rise in the prevalence of allergic diseases, and novel efficient therapeutic approaches are still needed to alleviate disease burden. Prostaglandin D2 (PGD2) has emerged as a central inflammatory lipid mediator associated with increased migration, activation and survival of leukocytes in various allergy-associated disorders. In the periphery, the hematopoietic PGD synthase (hPGDS) acts downstream of the arachidonic acid/COX pathway catalysing the isomerisation of PGH2 to PGD2, which makes it an interesting target to treat allergic inflammation. Although much effort has been put into developing efficient hPGDS inhibitors, no compound has made it to the market yet, which indicates that more light needs to be shed on potential PGD2 sources and targets to determine which particular condition and patient will benefit most and thereby improve therapeutic efficacy. In this review, we want to revisit current knowledge about hPGDS function, expression in allergy-associated cell types and their contribution to PGD2 levels as well as beneficial effects of hPGDS inhibition in allergic asthma, rhinitis, atopic dermatitis, food allergy, gastrointestinal allergic disorders and anaphylaxis.

Anticalin® Proteins as Therapeutic Agents in Human Diseases.

Anticalin proteins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies. Anticalin molecules are generated by combinatorial design from natural lipocalins, which are abundant plasma proteins in humans, and reveal a simple, compact fold dominated by a central ß-barrel, supporting four structurally variable loops that form a binding site. Reshaping of this loop region results in Anticalin proteins that can recognize and tightly bind a wide range of medically relevant targets, from small molecules to peptides and proteins, as validated by X-ray structural analysis. Their robust format allows for modification in several ways, both as fusion proteins and by chemical conjugation, for example, to tune plasma half-life. Antagonistic Anticalin therapeutics have been developed for systemic administration (e.g., PRS-080: anti-hepcidin) or pulmonary delivery (e.g. PRS-060/AZD1402: anti-interleukin [IL]-4-Rα). Moreover, Anticalin proteins allow molecular formatting as bi- and even multispecific fusion proteins, especially in combination with antibodies that provide a second specificity. For example, PRS-343, which has recently entered clinical-stage development, combines an agonistic Anticalin targeting the costimulatory receptor 4-1BB with an antibody directed against the cancer antigen human epidermal growth factor receptor 2 (HER2), thus offering a novel treatment option in immuno-oncology.

L-PGDS Mediates Vagus Nerve Stimulation-Induced Neuroprotection in a Rat Model of Ischemic Stroke by Suppressing the Apoptotic Response.

The role of lipocalin prostaglandin D2 synthase (L-PGDS) in brain ischemia has not been fully clarified to date. Vagus nerve stimulation (VNS) protects against cerebral ischemia/reperfusion (I/R) injury, but the mechanisms involved need further exploration. This study investigated the role of L-PGDS in cerebral I/R and whether this process was involved in the mechanism of VNS-mediated neuroprotection. Male Sprague-Dawley rats were pretreated with a lentiviral vector (LV) through intracerebroventricular injection, followed by middle cerebral artery occlusion (MCAO) and VNS treatment. The expression of L-PGDS in the peri-infarct cortex was examined. The localization of L-PGDS was determined using double immunofluorescence staining. Neurologic scores, infarct volume and neuronal apoptosis were evaluated at 24 h after reperfusion. The expression of apoptosis-related molecules was measured by western blot analysis. The expression of L-PGDS in the peri-infarct cortex increased at 12 h, reached a peak at 24 h after reperfusion, and lasted up to 3 days. VNS treatment further enhanced the expression of L-PGDS following ischemic stroke. L-PGDS was mainly expressed in neurons in the peri-infarct cortex. I/R rats treated with VNS showed better neurological deficit scores, reduced infarct volume, and decreased neuronal apoptosis as indicated by the decreased levels of Bax and cleaved caspase-3 as well as increased levels of Bcl-2. Strikingly, the beneficial effects of VNS were weakened after L-PGDS down-regulation. In general, our results suggest that L-PGDS is a potential mediator of VNS-induced neuroprotection against I/R injury.

High-affinity Anticalins with aggregation-blocking activity directed against the Alzheimer ß-amyloid peptide.

Amyloid beta (Aß) peptides, in particular Aß42 and Aß40, exert neurotoxic effects and their overproduction leads to amyloid deposits in the brain, thus constituting an important biomolecular target for treatments of Alzheimer's disease (AD). We describe the engineering of cognate Anticalins as a novel type of neutralizing protein reagent based on the human lipocalin scaffold. Phage display selection from a genetic random library comprising variants of the human lipocalin 2 (Lcn2) with mutations targeted at 20 exposed amino acid positions in the four loops that form the natural binding site was performed using both recombinant and synthetic target peptides and resulted in three different Anticalins. Biochemical characterization of the purified proteins produced by periplasmic secretion in Escherichia coli revealed high folding stability in a monomeric state, with Tm values ranging from 53.4°C to 74.5°C, as well as high affinities for Aß40, between 95 pM and 563 pM, as measured by real-time surface plasmon resonance analysis. The central linear VFFAED epitope within the Aß sequence was mapped using a synthetic peptide array on membranes and was shared by all three Anticalins, despite up to 13 mutual amino acid differences in their binding sites. All Anticalins had the ability-with varying extent-to inhibit Aß aggregation in vitro according to the thioflavin-T fluorescence assay and, furthermore, they abolished Aß42-mediated toxicity in neuronal cell culture. Thus, these Anticalins provide not only useful protein reagents to study the molecular pathology of AD but they also show potential as alternative drug candidates compared with antibodies.

Iron depletion strategy for targeted cancer therapy: utilizing the dual roles of neutrophil gelatinase-associated lipocalin protein.

Decreasing iron uptake and increasing iron efflux may result in cell death by oxidative inactivation of vital enzymes. Applying the dual function of neutrophil gelatinase-associated lipocalin (NGAL) could achieve the goal of iron depletion in the cancer cells. Tyr106, Lys125 or Lys134 was the key binding site for NGAL protein to sequester iron-chelating siderophores. In this study, we employed all bioactive peptides in peptide databank to dock with the siderophore-binding sites of NGAL protein by virtual screening. In addition, we performed molecular dynamics (MD) simulation to observe the molecular character and structural variation of ligand-protein interaction. Glu-Glu-Lys-Glu (EEKE), Glu-Glu-Asp-Cys-Lys (EEDCK), and Gly-Glu-Glu-Cys-Asp (GEECD) were selected preliminarily by rigorous scoring functions for further investigation. GEECD was excluded due to higher binding total energy than the others. Moreover, we also excluded EEKE due to larger influence to the stability of binding residues by the information of root mean square fluctuation (RMSF) and principal component analysis (PCA). Thus, we suggested that EEDCK was the potential bioactive peptide which had been proved to inhibit malignant cells for targeted cancer therapy. Graphical Abstract Perspective drug design of occupying the siderophore-binding sites of NGAL outside the cell temporarily by a potential short peptide until NGAL enters into the cell, and releasing the siderophore-binding sites inside the cell.

Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class.

Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood-retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.

Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

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.

Multimodality multiparametric imaging of early tumor response to a novel antiangiogenic therapy based on anticalins.

Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [18F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = -0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy.

A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity.

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be "fit for purpose" for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients.

Anticalins small engineered binding proteins based on the lipocalin scaffold.

Anticalins are a novel class of small, robust proteins with designed ligand-binding properties derived from the natural lipocalin scaffold. Due to their compact molecular architecture, comprising a single polypeptide chain, they provide several benefits as protein therapeutics, such as high target specificity, good tissue penetration, low immunogenicity, tunable plasma half-life, efficient Escherichia coli expression, and suitability for furnishing with additional effector functions via genetic fusion or chemical conjugation. The lipocalins are a widespread family of proteins that naturally serve in many organisms, including humans, for the transport, storage, or sequestration of small biological compounds like vitamins and hormones. Their fold is dominated by an eight-stranded antiparallel ß-barrel, which is open to the solvent at one end. There, four loops connect the ß-strands in a pairwise manner and, altogether, they form the entry to a ligand-binding site. This loop region can be engineered via site-directed random mutagenesis in combination with genetic library selection techniques to yield "Anticalins" with exquisite specificities-and down to picomolar affinities-for prescribed molecular targets of either hapten or antigen type. Several Anticalins directed against medically relevant disease targets have been successfully engineered and can be applied, for example, for the blocking of soluble signaling factors or cell surface receptors or for tissue-specific drug targeting. While natural lipocalins were already subject to clinical studies in the past, a first Anticalin has completed Phase I trials in 2011, thus paving the way for the broad application of Anticalins as a promising novel class of biopharmaceuticals.