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Iron is an essential nutrient for microbes, and many pathogenic bacteria depend on siderophores to obtain iron. The mammalian innate immunity protein lipocalin 2 (Lcn2; also known as neutrophil gelatinase-associated lipocalin, 24p3, or siderocalin) binds the siderophore carboxymycobactin, an essential component of the iron acquisition apparatus of mycobacteria. Here we show that Lcn2 suppressed growth of Mycobacterium avium in culture, and M. avium induced Lcn2 production from mouse macrophages. Lcn2 also had elevated levels and initially limited the growth of M. avium in the blood of infected mice but did not impede growth in tissues and during long-term infections. M. avium is an intracellular pathogen. Subcellular imaging of infected macrophages revealed that Lcn2 trafficked to lysosomes separate from M. avium, whereas transferrin was efficiently transported to the mycobacteria. Thus, mycobacteria seem to reside in the Rab11(+) endocytic recycling pathway, thereby retaining access to nutrition and avoiding endocytosed immunoproteins like Lcn2. [ABSTRACT FROM AUTHOR]

Mesothelin (MSLN) is a cell-surface glycoprotein expressed at low levels on normal mesothelium but overexpressed in many cancers. Mesothelin has been implicated to play role/s in cell adhesion and multiple signaling pathways. Mucin-16/CA125 is an enormous cell-surface glycoprotein, also normally expressed on mesothelium and implicated in the progression and metastasis of several cancers, and directly binds mesothelin. However, the precise biological function/s of mesothelin and mucin-16/CA125 remain mysterious. We report protein engineering and recombinant production, qualitative and quantitative binding studies, and a crystal structure determination elucidating the molecular-level details governing recognition of mesothelin by mucin-16/CA125. The interface is small, consistent with the ∼micromolar binding constant and is free of glycan-mediated interactions. Sequence comparisons and modeling suggest that multiple mucin-16/CA125 modules can interact with mesothelin through comparable interactions, potentially generating a high degree of avidity at the cell surface to overcome the weak affinity, with implications for functioning and therapeutic interventions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Targeted alpha therapy (TAT) pairs the specificity of antigen targeting with the lethality of alpha particles to eradicate cancerous cells. Actinium-225 [ 225 Ac; t 1/2  = 9.920(3) days] is an alpha-emitting radioisotope driving the next generation of TAT radiopharmaceuticals. Despite promising clinical results, a fundamental understanding of Ac coordination chemistry lags behind the rest of the Periodic Table due to its limited availability, lack of stable isotopes, and inadequate systems poised to probe the chemical behavior of this radionuclide. In this work, we demonstrate a platform that combines an 8-coordinate synthetic ligand and a mammalian protein to characterize the solution and solid-state behavior of the longest-lived Ac isotope, 227 Ac [t 1/2  = 21.772(3) years]. We expect these results to direct renewed efforts for 225 Ac-TAT development, aid in understanding Ac coordination behavior relative to other +3 lanthanides and actinides, and more broadly inform this element's position on the Periodic Table., (© 2024. The Author(s).)

NKG2D and its ligands are critical regulators of protective immune responses controlling infections and cancer, defining a crucial immune signaling axis. Current therapeutic efforts targeting this axis almost exclusively aim at enhancing NKG2D-mediated effector functions. However, this axis can drive disease processes when dysregulated, in particular, driving stem-like cancer cell reprogramming and tumorigenesis through receptor/ligand self-stimulation on tumor cells. Despite complexities with its structure and biology, we developed multiple novel engineered proteins that functionally serve as axis-blocking NKG2D "decoys" and report biochemical, structural, in vitro , and in vivo evaluation of their functionality., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Roland 10.13039/100014345Strong reports financial support was provided by 10.13039/100000060National Institute of Allergy and Infectious Diseases. Roland 10.13039/100014345Strong reports financial support was provided by 10.13039/100000002National Institutes of Health. Roland 10.13039/100014345Strong reports financial support was provided by 10.13039/100000054National Cancer Institute. Roland 10.13039/100014345Strong reports financial support was provided by US 10.13039/100000015Department of Energy. Roland 10.13039/100014345Strong reports financial support was provided by 10.13039/100001906Washington Research Foundation. Roland Strong has patent NKG2D DECOYS issued to Fred Hutchinson Cancer Center. Veronika Groh has patent NKG2D DECOYS issued to Fred Hutchinson Cancer Center. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

The recurrence of malignancy after hematopoietic cell transplantation (HCT) is the primary cause of transplantation failure. The NKG2D axis is a powerful pathway for antitumor responses, but its role in the control of malignancy after HCT is not well-defined. We tested the hypothesis that gene variation of the NKG2D receptor and its ligands MICA and MICB affect relapse and survival in 1629 patients who received a haploidentical HCT for the treatment of a malignant blood disorder. Patients and donors were characterized for MICA residue 129, the exon 5 short tandem repeat (STR), and MICB residues 52, 57, 98, and 189. Donors were additionally defined for the presence of NKG2D residue 72. Mortality was higher in patients with MICB-52Asn relative to those with 52Asp (hazard ratio [HR], 1.83; 95% confidence interval [CI], 1.24-2.71; P = .002) and lower in those with MICA-STR mismatch than in those with STR match (HR, 0.66; 95% CI, 0.54-0.79; P = .00002). Relapse was lower with NKG2D-72Thr donors than with 72Ala donors (relapse HR, 0.57; 95% CI, 0.35-0.91; P = .02). The protective effects of patient MICB-52Asp with donor MICA-STR mismatch and NKG2D-72Thr were enhanced when all 3 features were present. The NKG2D ligand/receptor pathway is a transplantation determinant. The immunobiology of relapse is defined by the concerted effects of MICA, MICB, and NKG2D germ line variation. Consideration of NKG2D ligand/receptor pairings may improve survival for future patients., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The mammalian protein siderocalin binds bacterial siderophores and their iron complexes through cation-π and electrostatic interactions, but also displays high affinity for hydroxypyridinone complexes of trivalent lanthanides and actinides. In order to circumvent synthetic challenges, the use of siderocalin-antibody fusion proteins is explored herein as an alternative targeting approach for precision delivery of trivalent radiometals. We demonstrate the viability of this approach in vivo , using the theranostic pair 90 Y (β - , t 1/2 = 64 h)/ 86 Y (β + , t 1/2 = 14.7 h) in a SKOV-3 xenograft mouse model. Ligand radiolabeling with octadentate hydroxypyridinonate 3,4,3-LI(1,2-HOPO) and subsequent protein binding were achieved at room temperature. The results reported here suggest that the rapid non-covalent binding interaction between siderocalin fusion proteins and the negatively charged Y(iii)-3,4,3-LI(1,2-HOPO) complexes could enable purification-free, cold-kit labeling strategies for the application of therapeutically relevant radiometals in the clinic., Competing Interests: RJA and RKS are listed as inventors on patent applications filed by the Lawrence Berkeley National Laboratory (LBNL) and the Fred Hutchinson Cancer Center, describing inventions related to the research results presented here. The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

The discovery and characterization of antigen-specific CD8 + T cell clonotypes typically involves the labor-intensive synthesis and construction of peptide-MHC tetramers. We adapt single-chain trimer (SCT) technologies into a high throughput platform for pMHC library generation, showing that hundreds can be rapidly prepared across multiple Class I HLA alleles. We use this platform to explore the impact of peptide and SCT template mutations on protein expression yield, thermal stability, and functionality. SCT libraries were an efficient tool for identifying T cells recognizing commonly reported viral epitopes. We then construct SCT libraries to capture SARS-CoV-2 specific CD8 + T cells from COVID-19 participants and healthy donors. The immunogenicity of these epitopes is validated by functional assays of T cells with cloned TCRs captured using SCT libraries. These technologies should enable the rapid analyses of peptide-based T cell responses across several contexts, including autoimmunity, cancer, or infectious disease., (© 2023. The Author(s).)

MHC class I " single-chain trimer " molecules, coupling MHC heavy chain, β 2 -microglobulin, and a specific peptide into a single polypeptide chain, are widely used in research. To more fully understand caveats associated with this design that may affect its use for basic and translational studies, we evaluated a set of engineered single-chain trimers with combinations of stabilizing mutations across eight different classical and non-classical human class I alleles with 44 different peptides, including a novel human/murine chimeric design. While, overall, single-chain trimers accurately recapitulate native molecules, care was needed in selecting designs for studying peptides longer or shorter than 9-mers, as single-chain trimer design could affect peptide conformation. In the process, we observed that predictions of peptide binding were often discordant with experiment and that yields and stabilities varied widely with construct design. We also developed novel reagents to improve the crystallizability of these proteins and confirmed novel modes of peptide presentation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Finton, Rupert, Friend, Dinca, Lovelace, Buerger, Rusnac, Foote-McNabb, Chour, Heath, Campbell, Pierce and Strong.)

Opioid-related fatal overdoses have reached epidemic proportions. Because existing treatments for opioid use disorders offer limited long-term protection, accelerating the development of newer approaches is critical. Monoclonal antibodies (mAbs) are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier, thus preventing or reversing opioid toxicity. We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine. To determine their binding mechanism, we used X-ray crystallography to solve the structures of mAbs bound to their respective targets, to 2.2 Å resolution or higher. Structural analysis showed a critical convergent hydrogen bonding mode that is dependent on a glutamic acid residue in the mAbs' heavy chain and a tertiary amine of the ligand. Characterizing drug-mAb complexes represents a significant step toward rational antibody engineering and future manufacturing activities to support clinical evaluation., Competing Interests: Declaration of interests M. Pravetoni, D.H., and C.B. are inventors of provisional and non-provisional patent applications covering the anti-fentanyl mAbs described herein., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Cystine-dense peptides (CDPs) are a miniprotein class that can drug difficult targets with high affinity and low immunogenicity. Tools for their design, however, are not as developed as those for small-molecule and antibody drugs. CDPs have diverse taxonomic origins, but structural characterization is lacking. Here, we adapted Iterative Threading ASSEmbly Refinement (I-TASSER) and Rosetta protein modeling software for structural prediction of 4298 CDP scaffolds and performed in silico prescreening for CDP binders to targets of interest. Mammalian display screening of a library of docking-enriched, methionine and tyrosine scanned (DEMYS) CDPs against PD-L1 yielded binders from four distinct CDP scaffolds. One was affinity-matured, and cocrystallography yielded a high-affinity ( K D = 202 pM) PD-L1-binding CDP that competes with PD-1 for PD-L1 binding. Its subsequent incorporation into a CD3-binding bispecific T cell engager produced a molecule with pM-range in vitro T cell killing potency and which substantially extends survival in two different xenograft tumor-bearing mouse models. Both in vitro and in vivo, the CDP-incorporating bispecific molecule outperformed a comparator antibody-based molecule. This CDP modeling and DEMYS technique can accelerate CDP therapeutic development.

Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND 50 ) geometric mean titers (GMTs) that were highly correlated (Pearson r  = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND 50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD ( r  = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG ( r  = 0.46 to 0.73). ND 80 GMTs mirrored ND 50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Conventional immunoprecipitation/mass spectroscopy identification of HLA-restricted peptides remains the purview of specializing laboratories, due to the complexity of the methodology, and requires computational post-analysis to assign peptides to individual alleles when using pan-HLA antibodies. We have addressed these limitations with ARTEMIS: a simple, robust, and flexible platform for peptide discovery across ligandomes, optionally including specific proteins-of-interest, that combines novel, secreted HLA-I discovery reagents spanning multiple alleles, optimized lentiviral transduction, and streamlined affinity-tag purification to improve upon conventional methods. This platform fills a middle ground between existing techniques: sensitive and adaptable, but easy and affordable enough to be widely employed by general laboratories. We used ARTEMIS to catalog allele-specific ligandomes from HEK293 cells for seven classical HLA alleles and compared results across replicates, against computational predictions, and against high-quality conventional datasets. We also applied ARTEMIS to identify potentially useful, novel HLA-restricted peptide targets from oncovirus oncoproteins and tumor-associated antigens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Finton, Brusniak, Jones, Lin, Fioré-Gartland, Brock, Gafken and Strong.)

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (K D as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development., Competing Interests: Competing Interests Z.R.C., E.J.G., C.Y., G.H., A.D.S., A.J.M., R.K.S., and J.M.O. are listed as authors on patent filings related to technology described in this work. Z.R.C. and G.P.S. have a paid consulting relationship with Blaze Bioscience, Inc. J.M.O. is a founder and shareholder of Blaze Bioscience, Inc. The remaining authors declare no competing financial interests., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies., (© 2019 Bancroft et al.)

Bacterial flagellin is a well-known agonist of the innate immune system that induces proinflammatory responses through the TLR5 and Naip5/6 recognition pathways. Several clinical trials investigating flagellin fusion proteins have demonstrated promising results for inducing protective immunity toward influenza virus, which has been largely attributed to flagellin's ability to activate TLR5. Our laboratory previously demonstrated that the Salmonella enterica serovar Typhimurium flagellin protein, FliC, induces Ab responses in mice through a third pathway that is independent of TLR5, Casp1/11, and MyD88. In this study, we further define the structural features of FliC that contribute to this unknown third pathway. By destroying the Naip5/6 and TLR5 recognition sites, we demonstrate that neither were required for the TLR5-, inflammasome- and MyD88-independent Ab responses toward FliC. In contrast, deletion of FliC's D3 or D0/D1 domains eliminated primary anti-flagellin Ab responses. For optimal primary and secondary anti-flagellin Ab responses we show that TLR5, inflammasome recognition, and the D3 domain of FliC are essential for flagellin's robust immunogenicity. Our data demonstrate that the D3 domain of FliC influences immunogenicity independent of the known innate recognition sites in the D0/D1 domains to augment Ab production. Our results suggest full-length FliC is critical for optimal immunogenicity and Ab responses in flagellin-based vaccines., (Copyright © 2019 The Authors.)

Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration - including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism., Competing Interests: The authors declare no conflict of interest with respect to this publication., (© 2019 Published by Elsevier Inc.)

Purpose HLA mismatching increases mortality after unrelated donor hematopoietic cell transplantation. The role of the patient's germline variation on survival is not known. Patients and Methods We previously identified 12 single nucleotide polymorphisms within the HLA region as markers of transplantation determinants and tested these in an independent cohort of 1,555 HLA-mismatched unrelated transplants. Linkage disequilibrium mapping across class II identified candidate susceptibility features. The candidate gene was confirmed in an independent cohort of 3,061 patients. Results Patient rs429916AA/AC was associated with increased transplantation-related mortality compared with rs429916CC (hazard ratio [HR], 1.39; 95% CI, 1.12 to 1.73; P = .003); rs429916A positivity was a proxy for DOA*01:01:05. Mortality increased with one (HR, 1.17; 95% CI, 1.0 to 1.36; P = .05) and two (HR, 2.51; 95% CI, 1.41 to 4.45; P = .002) DOA*01:01:05 alleles. HLA-DOA*01:01:05 was a proxy for HLA-DRB1 alleles encoding FEY ( P < 10E -15 ) and FDH ( P < 10E -15 ) amino acid substitutions at residues 26/28/30 that influence HLA-DRβ peptide repertoire. FEY- and FDH-positive alleles were positively associated with rs429916A ( P < 10E -15 ); FDY-positive alleles were negatively associated. Mortality was increased with FEY (HR, 1.66; 95% CI, 1.29 to 2.13; P = .00008) and FDH (HR, 1.40; 95% CI, 1.02 to 1.93; P = .04), whereas FDY was protective (HR, 0.88; 95% CI, 0.78 to 0.98; P = .02). Of the three candidate motifs, FEY was validated as the susceptibility determinant for mortality (HR, 1.29; 95% CI, 1.00 to 1.67; P = .05). Although FEY was found frequently among African and Hispanic Americans, it increased mortality independently of ancestry. Conclusion Patient germline HLA-DRB1 alleles that encode amino acid substitutions that influence the peptide repertoire of HLA-DRβ predispose to increased death after transplantation. Patient germline variation informs transplantation outcomes across US populations and may provide a means to reduce risks for high-risk patients through pretransplantation screening and evaluation.

In the original version of this Article the colour key for the amino acid enrichment score was inadvertently omitted from the lower panel of Figure 5b during the production process. This has now been corrected in the PDF and HTML versions of the Article.

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria. Many showed extreme resistance to temperature, proteolysis and/or reduction, and all displayed inhibitory activity against at least 1 of 20 ion channels tested, thus confirming their biological functionality. Crystal structures of 12 confirmed proper cystine topology and the utility of crystallography to study these molecules but also highlighted the need for rational classification. Previous categorization attempts have focused on limited subsets featuring distinct motifs. Here we present a global definition, classification and analysis of >700 structures of cystine-dense peptides, providing a unifying framework for these molecules.

Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability. We demonstrate the platform's capabilities by identifying a cystine-dense peptide capable of inhibiting the YAP:TEAD interaction at the heart of the oncogenic Hippo pathway, and possessing the potency and stability necessary for consideration as a drug development candidate. This platform provides the opportunity to screen cystine-dense peptides with drug-like qualities against targets that are implicated for the treatment of diseases, but are poorly suited for conventional approaches.

Berkelium (Bk) has been predicted to be the only transplutonium element able to exhibit both +III and +IV oxidation states in solution, but evidence of a stable oxidized Bk chelate has so far remained elusive. Here we describe the stabilization of the heaviest 4+ ion of the periodic table, under mild aqueous conditions, using a siderophore derivative. The resulting Bk(IV) complex exhibits luminescence via sensitization through an intramolecular antenna effect. This neutral Bk(IV) coordination compound is not sequestered by the protein siderocalin-a mammalian metal transporter-in contrast to the negatively charged species obtained with neighbouring trivalent actinides americium, curium and californium (Cf). The corresponding Cf(III)-ligand-protein ternary adduct was characterized by X-ray diffraction analysis. Combined with theoretical predictions, these data add significant insight to the field of transplutonium chemistry, and may lead to innovative Bk separation and purification processes.

Cancer cells may co-opt the NKG2D lymphocyte receptor to complement the presence of its ligands for autonomous stimulation of oncogenic signaling. Previous studies raise the possibility that cancer cell NKG2D may induce high malignancy traits, but its full oncogenic impact is unknown. Using epithelial ovarian cancer as model setting, we show here that ex vivo NKG2D + cancer cells have stem-like capacities, and provide formal in vivo evidence linking NKG2D stimulation with the development and maintenance of these functional states. NKG2D + ovarian cancer cell populations harbor substantially greater capacities for self-renewing in vitro sphere formation and in vivo tumor initiation in immunodeficient (NOD scid gamma) mice than NKG2D - controls. Sphere formation and tumor initiation are impaired by NKG2D silencing or ligand blockade using antibodies or a newly designed pan ligand-masking NKG2D multimer. In further support of pathophysiological significance, a prospective study of 47 high-grade serous ovarian cancer cases revealed that the odds of disease recurrence were significantly greater and median progression-free survival rates higher among patients with above and below median NKG2D + cancer cell frequencies, respectively. Collectively, our results define cancer cell NKG2D as an important regulator of tumor initiation in ovarian cancer and presumably other malignancies and thus challenge current efforts in immunotherapy aimed at enhancing NKG2D function., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeted α therapy holds tremendous potential as a cancer treatment: it offers the possibility of delivering a highly cytotoxic dose to targeted cells while minimizing damage to surrounding healthy tissue. The metallic α-generating radioisotopes 225 Ac and 227 Th are promising radionuclides for therapeutic use, provided adequate chelation and targeting. Here we demonstrate a new chelating platform composed of a multidentate high-affinity oxygen-donating ligand 3,4,3-LI(CAM) bound to the mammalian protein siderocalin. Respective stability constants log β 110 = 29.65 ± 0.65, 57.26 ± 0.20, and 47.71 ± 0.08, determined for the Eu III (a lanthanide surrogate for Ac III ), Zr IV , and Th IV complexes of 3,4,3-LI(CAM) through spectrophotometric titrations, reveal this ligand to be one of the most powerful chelators for both trivalent and tetravalent metal ions at physiological pH. The resulting metal-ligand complexes are also recognized with extremely high affinity by the siderophore-binding protein siderocalin, with dissociation constants below 40 nM and tight electrostatic interactions, as evidenced by X-ray structures of the protein:ligand:metal adducts with Zr IV and Th IV . Finally, differences in biodistribution profiles between free and siderocalin-bound 238 Pu IV -3,4,3-LI(CAM) complexes confirm in vivo stability of the protein construct. The siderocalin:3,4,3-LI(CAM) assembly can therefore serve as a "lock" to consolidate binding to the therapeutic 225 Ac and 227 Th isotopes or to the positron emission tomography emitter 89 Zr, independent of metal valence state.

Iron overload damages many organs. Unfortunately, therapeutic iron chelators also have undesired toxicity and may deliver iron to microbes. Here we show that a mutant form (K3Cys) of endogenous lipocalin 2 (LCN2) is filtered by the kidney but can bypass sites of megalin-dependent recapture, resulting in urinary excretion. Because K3Cys maintains recognition of its cognate ligand, the iron siderophore enterochelin, this protein can capture and transport iron even in the acidic conditions of urine. Mutant LCN2 strips iron from transferrin and citrate, and delivers it into the urine. In addition, it removes iron from iron overloaded mice, including models of acquired (iron-dextran or stored red blood cells) and primary (Hfe -/- ) iron overload. In each case, the mutants reduce redox activity typical of non-transferrin-bound iron. In summary, we present a non-toxic strategy for iron chelation and urinary elimination, based on manipulating an endogenous protein:siderophore:iron clearance pathway., Competing Interests: J.B. and A.Q. declare patent applications with Columbia University for the use of LCN2 (Scn or NGAL). R.K.S., P.B.R. and R.J.A. declare patent applications with Fred Hutchinson Cancer Research Center for the use of Scn (NGAL). All the other authors declare no competing financial interest.

Based on previous findings supporting HLA-F as a ligand for KIR3DL2 and KIR2DS4, we investigated the potential for MHC-I open conformers (OCs) as ligands for KIR3DS1 and KIR3DL1 through interactions measured by surface plasmon resonance. These measurements showed physical binding of KIR3DS1 but not KIR3DL1 with HLA-F and other MHC-I OC while also confirming the allotype specific binding of KIR3DL1 with MHC-I peptide complex. Concordant results were obtained with biochemical pull-down from cell lines and biochemical heterodimerization experiments with recombinant proteins. In addition, surface binding of HLA-F and KIR3DS1 to native and activated NK and T cells was coincident with specific expression of the putative ligand or receptor. A functional response of KIR3DS1 was indicated by increased granule exocytosis in activated cells incubated with HLA-F bound to surfaces. The data extend a model for interaction between MHC-I open conformers and activating KIR receptors expressed during an inflammatory response, potentially contributing to communication between the innate and adaptive immune response., Competing Interests: The authors have declared that no competing interests exist.

VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications.

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.

Background: CD28 signal blockade following T cell receptor activation is under intense investigation as a tolerance-inducing therapy for transplantation. Our goal is to produce a CD28-specific reagent as a therapy for the prevention of graft rejection and graft-versus-host disease in the canine model of allogeneic hematopoietic cell transplantation (HCT)., Methods: We infused a monoclonal mouse anti-canine CD28 antibody (1C6 mAb) into four dogs and a fragment of antigen-binding (1C6 Fab) into two dogs. Pharmacokinetics, pathology, cytokine release, and the crystal structure of 1C6 Fv were evaluated., Results: Within an hour of an IV injection of the 1C6 mAb, the dogs became leukopenic and developed a steroid-refractory cytokine storm. Two of the dogs developed high fevers, one experienced diffuse alveolar hemorrhage, and another developed gastrointestinal hemorrhage. The cytokine storm was characterized by elevated plasma levels of MCP-1, IP-10, IL-10, IL-6, and TNF-α. In addition, one dog showed elevated levels of IL-2, IL-8, and IL-18. In contrast, infusion of 1C6 Fab was well tolerated without any side effects. Dry-coating 1C6 mAb onto tissue culture plates induced CD3-independent proliferation and TNF-alpha production. Crystal structure analysis revealed that 1C6 binds to canine CD28 in a manner different than previously reported for conventional agonistic or superagonistic antibodies., Conclusions: These results indicate that dogs and humans develop a similar cytokine storm following infusion ofanti-CD28 mAb, providing an appropriate large animal for further study. 1C6 Fab warrants evaluation as a tolerance-inducing reagent in the canine model of allogeneic HCT.

Competition for iron is a critical component of successful bacterial infections, but the underlying in vivo mechanisms are poorly understood. We have previously demonstrated that lipocalin 2 (LCN2) is an innate immunity protein that binds to bacterial siderophores and starves them for iron, thus representing a novel host defense mechanism to infection. In the present study we show that LCN2 is secreted by the urinary tract mucosa and protects against urinary tract infection (UTI). We found that LCN2 was expressed in the bladder, ureters, and kidneys of mice subject to UTI. LCN2 was protective with higher bacterial numbers retrieved from bladders of Lcn2-deficient mice than from wild-type mice infected with the LCN2-sensitive Escherichia coli strain H9049. Uropathogenic E. coli mutants in siderophore receptors for salmochelin, aerobactin, or yersiniabactin displayed reduced fitness in wild-type mice, but not in mice deficient of LCN2, demonstrating that LCN2 imparts a selective pressure on bacterial growth in the bladder. In a human cohort of women with recurrent E. coli UTIs, urine LCN2 levels were associated with UTI episodes and with levels of bacteriuria. The number of siderophore systems was associated with increasing bacteriuria during cystitis. Our data demonstrate that LCN2 is secreted by the urinary tract mucosa in response to uropathogenic E. coli challenge and acts in innate immune defenses as a colonization barrier that pathogens must overcome to establish infection., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.

Lipocalins are small extracellular proteins mostly described as lipid carriers. The Drosophila lipocalin NLaz (neural Lazarillo) modulates the IIS pathway and regulates longevity, stress resistance, and behavior. Here, we test whether a native hydrophobic pocket structure is required for NLaz to perform its functions. We use a point mutation altering the binding pocket (NLaz(L130R)) and control mutations outside NLaz binding pocket. Tryptophan fluorescence titration reveals that NLaz(L130R) loses its ability to bind ergosterol and the pheromone 7(z)-tricosene but retains retinoic acid binding. Using site-directed transgenesis in Drosophila, we test the functionality of the ligand binding-altered lipocalin at the organism level. NLaz-dependent life span reduction, oxidative stress and starvation sensitivity, aging markers accumulation, and deficient courtship are rescued by overexpression of NLaz(WT), but not of NLaz(L130R). Transcriptional responses to aging and oxidative stress show a large set of age-responsive genes dependent on the integrity of NLaz binding pocket. Inhibition of IIS activity and modulation of oxidative stress and infection-responsive genes are binding pocket-dependent processes. Control of energy metabolites on starvation appears to be, however, insensitive to the modification of the NLaz binding pocket.

Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1Rs and IL-18Rs, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. In this study, we define roles of known flagellin receptors and MyD88 in Ab responses generated toward flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype-specific Ab responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote Ab responses toward flagellin and coadministered OVA in C57BL/6 mice. We demonstrate IgG2c responses toward flagellin were TLR5 and inflammasome dependent; IgG1 was the dominant isotype and partially TLR5 and inflammasome dependent. Our data indicate a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5 and MyD88 dependent and caspase-1 independent. Unlike C57BL/6 mice, flagellin-immunized A/J mice induced codominant IgG1 and IgG2a responses. Furthermore, MyD88-independent, flagellin-induced Ab responses were even more pronounced in A/J MyD88(-/-) mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward coadministered OVA, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to Ab production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.

The human protein siderocalin (Scn) inhibits bacterial iron acquisition by binding catechol siderophores. Several pathogenic bacteria respond by making stealth siderophores that are not recognized by Scn. Fluvibactin and vibriobactin, respectively of Vibrio fluvialis and Vibrio cholerae , include an oxazoline adjacent to a catechol. This chelating unit binds iron either in a catecholate or a phenolate-oxazoline coordination mode. The latter has been suggested to make vibriobactin a stealth siderophore without directly identifying the coordination mode in relation to Scn binding. We use Scn binding assays with the two siderophores and two oxazoline-substituted analogs and the crystal structure of Fe-fluvibactin:Scn to show that the oxazoline does not prevent Scn binding; hence, vibriobactin is not a stealth siderophore. We show that the phenolate-oxazoline coordination mode is present at physiological pH and is not bound by Scn. However, Scn binding shifts the coordination to the catecholate mode and thereby inactivates this siderophore.

Lipocalins are a family of proteins characterized by a conserved eight-stranded β-barrel structure with a ligand-binding pocket. They perform a wide range of biological functions and this functional multiplicity must relate to the lipid partner involved. Apolipoprotein D (ApoD) and its insect homologues, Lazarillo (Laz) and neural Lazarillo (NLaz), share common ancestral functions like longevity, stress resistance and lipid metabolism regulation, coexisting with very specialized functions, like courtship behavior. Using tryptophan fluorescence titration, we screened the binding of 15 potential lipid partners for NLaz, ApoD and Laz and uncovered several novel ligands with apparent dissociation constants in the low micromolar range. Retinoic acid (RA), retinol, fatty acids and sphingomyelin are shared ligands. Sterols, however, showed a species-specific binding pattern: cholesterol did not show strong binding to human ApoD, whereas NLaz and Laz did bind ergosterol. Among the lipocalin-specific ligands, we found that ApoD selectively binds the endocannabinoid anandamide but not 2-acylglycerol, and that NLaz binds the pheromone 7-tricosene, but not 7,11-heptacosadiene or 11-cis-vaccenyl acetate. To test the functional relevance of lipocalin ligand binding at the cellular level, we analyzed the effect of ApoD, Laz and NLaz preloaded with RA on neuronal differentiation. Our results show that ApoD is necessary and sufficient to allow for RA differentiating activity. Both human ApoD and Drosophila NLaz successfully deliver RA to immature neurons, driving neurite outgrowth. We conclude that ApoD, NLaz and Laz bind selectively to a different but overlapping set of lipid ligands. This multispecificity can explain their varied physiological functions., (© 2013 FEBS.)

The broadly-neutralizing anti-HIV antibody 4E10 recognizes an epitope in the membrane-proximal external region of the HIV envelope protein gp41. Previous attempts to elicit 4E10 by vaccination with envelope-derived or reverse-engineered immunogens have failed. It was presumed that the ontogeny of 4E10-equivalent responses was blocked by inherent autoreactivity and exceptional polyreactivity. We generated 4E10 heavy-chain knock-in mice, which displayed significant B cell dysregulation, consistent with recognition of autoantigen/s by 4E10 and the presumption that tolerance mechanisms may hinder the elicitation of 4E10 or 4E10-equivalent responses. Previously proposed candidate 4E10 autoantigens include the mitochondrial lipid cardiolipin and a nuclear splicing factor, 3B3. However, using carefully-controlled assays, 4E10 bound only weakly to cardiolipin-containing liposomes, but also bound negatively-charged, non-cardiolipin-containing liposomes comparably poorly. 4E10/liposome binding was predominantly mediated by electrostatic interactions rather than presumed hydrophobic interactions. The crystal structure of 4E10 free of bound ligands showed a dramatic restructuring of the combining site, occluding the HIV epitope binding site and revealing profound flexibility, but creating an electropositive pocket consistent with non-specific binding of phospholipid headgroups. These results strongly suggested that antigens other than cardiolipin mediate 4E10 autoreactivity. Using a synthetic peptide library spanning the human proteome, we determined that 4E10 displays limited and focused, but unexceptional, polyspecificity. We also identified a novel autoepitope shared by three ER-resident inositol trisphosphate receptors, validated through binding studies and immunohistochemistry. Tissue staining with 4E10 demonstrated reactivity consistent with the type 1 inositol trisphosphate receptor as the most likely candidate autoantigen, but is inconsistent with splicing factor 3B3. These results demonstrate that 4E10 recognition of liposomes competes with MPER recognition and that HIV antigen and autoepitope recognition may be distinct enough to permit eliciting 4E10-like antibodies, evading autoimmunity through directed engineering. However, 4E10 combining site flexibility, exceptional for a highly-matured antibody, may preclude eliciting 4E10 by conventional immunization strategies.

Vaccine candidates for HIV-1 so far have not been able to elicit broadly neutralizing antibodies (bNAbs) although they express the epitopes recognized by bNAbs to the HIV envelope glycoprotein (Env). To understand whether and how Env immunogens interact with the predicted germline versions of known bNAbs, we screened a large panel (N:56) of recombinant Envs (from clades A, B and C) for binding to the germline predecessors of the broadly neutralizing anti-CD4 binding site antibodies b12, NIH45-46 and 3BNC60. Although the mature antibodies reacted with diverse Envs, the corresponding germline antibodies did not display Env-reactivity. Experiments conducted with engineered chimeric antibodies combining the mature and germline heavy and light chains, respectively and vice-versa, revealed that both antibody chains are important for the known cross-reactivity of these antibodies. Our results also indicate that in order for b12 to display its broad cross-reactivity, multiple somatic mutations within its VH region are required. A consequence of the failure of the germline b12 to bind recombinant soluble Env is that Env-induced B-cell activation through the germline b12 BCR does not take place. Our study provides a new explanation for the difficulties in eliciting bNAbs with recombinant soluble Env immunogens. Our study also highlights the need for intense efforts to identify rare naturally occurring or engineered Envs that may engage the germline BCR versions of bNAbs.

Natural killer (NK) cells are key components of innate immune responses, providing surveillance against cells undergoing tumorigenesis or infection, by viruses or internal pathogens. NK cells can directly eliminate compromised cells and regulate downstream responses of the innate and acquired immune systems through the release of immune modulators (cytokines, interferons). The importance of the role NK cells play in immune defense was demonstrated originally in herpes viral infections, usually mild or localized, which become severe and life threatening in NK-deficient patients . NK cell effector functions are governed by balancing opposing signals from a diverse array of activating and inhibitory receptors. Many NK receptors occur in paired activating and inhibitory isoforms and recognize major histocompatibility complex (MHC) class I proteins with varying degrees of peptide specificity. Structural studies have made considerable inroads into understanding the molecular mechanisms employed to broadly recognize multiple MHC ligands or specific pathogen-associated antigens and the strategies employed by viruses to thwart these defenses. Although many details of NK development, signaling, and integration remain mysterious, it is clear that NK receptors are key components of a system exquisitely tuned to sense any dysregulation in MHC class I expression, or the expression of certain viral antigens, resulting in the elimination of affected cells., (© 2012 John Wiley & Sons A/S.)

Bacteria use tight-binding, ferric-specific chelators called siderophores to acquire iron from the environment and from the host during infection; animals use proteins such as transferrin and ferritin to transport and store iron. Recently, candidate compounds that could serve endogenously as mammalian siderophore equivalents have been identified and characterized through associations with siderocalin, the only mammalian siderophore-binding protein currently known. Siderocalin, an antibacterial protein, acts by sequestering iron away from infecting bacteria as siderophore complexes. Candidate endogenous siderophores include compounds that only effectively transport iron as ternary complexes with siderocalin, explaining pleiotropic activities in normal cellular processes and specific disease states.