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1.
J Mol Biol ; 432(14): 3989-4009, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32304700

RESUMO

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 (KD 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.

2.
Sci Transl Med ; 12(533)2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132215

RESUMO

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.

3.
Immunohorizons ; 3(9): 422-432, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488506

RESUMO

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.


Assuntos
Flagelina/metabolismo , Vacinas contra Salmonella/imunologia , Salmonella typhi/fisiologia , Febre Tifoide/imunologia , Animais , Anticorpos Antibacterianos/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Flagelina/genética , Flagelina/imunologia , Humanos , Imunidade Humoral , Imunidade Inata , Camundongos , Camundongos Knockout , Microrganismos Geneticamente Modificados , Mutação/genética , Proteína Inibidora de Apoptose Neuronal/genética , Proteína Inibidora de Apoptose Neuronal/metabolismo , Domínios Proteicos/genética , Transdução de Sinais , Receptor 5 Toll-Like/genética
4.
J Exp Med ; 216(10): 2331-2347, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31345930

RESUMO

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.

5.
J Clin Oncol ; 36(24): 2524-2531, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-29902106

RESUMO

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-DRß 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-DRß 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.


Assuntos
Cadeias HLA-DRB1/genética , Transplante de Células-Tronco Hematopoéticas/mortalidade , Genótipo , Antígenos HLA/genética , Humanos , Polimorfismo de Nucleotídeo Único , Doadores não Relacionados
6.
Nat Commun ; 9(1): 1072, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523778

RESUMO

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.

7.
Nat Struct Mol Biol ; 25(3): 270-278, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29483648

RESUMO

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.


Assuntos
Cistina/química , Peptídeos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Células HEK293 , Humanos , Canais Iônicos/antagonistas & inibidores , Modelos Moleculares , Biossíntese Peptídica , Peptídeos/classificação , Peptídeos/farmacologia
8.
Nat Commun ; 8(1): 2244, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29269835

RESUMO

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.


Assuntos
Cistina/análise , Peptídeos/química , Peptídeos/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Descoberta de Drogas , Proteínas de Escherichia coli/química , Glicosilação , Humanos , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/química
9.
Nat Chem ; 9(9): 843-849, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28837177

RESUMO

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.

10.
Neoplasia ; 19(6): 471-482, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28499126

RESUMO

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.


Assuntos
Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos/administração & dosagem , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Subfamília K de Receptores Semelhantes a Lectina de Células NK/antagonistas & inibidores , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nat Commun ; 7: 12973, 2016 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-27796299

RESUMO

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.


Assuntos
Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , Lipocalina-2/genética , Lipocalina-2/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Inflamação , Quelantes de Ferro , Sobrecarga de Ferro/genética , Rim/metabolismo , Ligantes , Camundongos , Camundongos Transgênicos , Mutação , Oxirredução , Ligação Proteica , Sideróforos , Transferrina/metabolismo
12.
Inorg Chem ; 55(22): 11930-11936, 2016 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-27802058

RESUMO

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 225Ac and 227Th 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 ß110 = 29.65 ± 0.65, 57.26 ± 0.20, and 47.71 ± 0.08, determined for the EuIII (a lanthanide surrogate for AcIII), ZrIV, and ThIV 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 ZrIV and ThIV. Finally, differences in biodistribution profiles between free and siderocalin-bound 238PuIV-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 225Ac and 227Th isotopes or to the positron emission tomography emitter 89Zr, independent of metal valence state.


Assuntos
Quelantes/química , Complexos de Coordenação/química , Proteínas/química , Radioterapia/métodos , Tório/química , Zircônio/química , Animais , Complexos de Coordenação/farmacocinética , Feminino , Ligantes , Camundongos , Modelos Químicos , Distribuição Tecidual
13.
PLoS One ; 11(9): e0163297, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27649529

RESUMO

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.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/metabolismo , Receptores KIR3DS1/metabolismo , Humanos , Células Matadoras Naturais/citologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína
14.
Nat Commun ; 7: 10618, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26907590

RESUMO

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.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos B/imunologia , Técnicas de Introdução de Genes , HIV-1/genética , Humanos , Imunização , Fragmentos Fab das Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
17.
Proc Natl Acad Sci U S A ; 112(33): 10342-7, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26240330

RESUMO

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.


Assuntos
Elementos da Série Actinoide/química , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas/química , Elementos da Série Actinoide/farmacocinética , Quelantes/química , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Íons , Cinética , Elementos da Série dos Lantanídeos , Ligantes , Luminescência , Metais/química , Conformação Molecular , Centrais Nucleares , Fotoquímica , Ligação Proteica , Liberação Nociva de Radioativos , Espectrofotometria , Eletricidade Estática , Difração de Raios X
18.
PLoS Pathog ; 11(5): e1004857, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25974133

RESUMO

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.


Assuntos
Adesinas de Escherichia coli/metabolismo , Anticorpos Monoclonais/imunologia , Aderência Bacteriana , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Escherichia coli Uropatogênica/metabolismo , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares
19.
Transplant Direct ; 1(2)2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25988188

RESUMO

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.

20.
J Immunol ; 193(12): 6081-9, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25398327

RESUMO

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.


Assuntos
Proteínas da Fase Aguda/genética , Infecções Bacterianas/genética , Lipocalinas/genética , Proteínas Proto-Oncogênicas/genética , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Proteínas da Fase Aguda/metabolismo , Adolescente , Adulto , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/metabolismo , Infecções Bacterianas/patologia , Carga Bacteriana , Cistite/genética , Cistite/imunologia , Cistite/metabolismo , Cistite/microbiologia , Modelos Animais de Doenças , Escherichia coli , Feminino , Expressão Gênica , Humanos , Ferro/metabolismo , Lipocalina-2 , Lipocalinas/metabolismo , Camundongos , Pessoa de Meia-Idade , Membrana Mucosa/imunologia , Membrana Mucosa/metabolismo , Membrana Mucosa/patologia , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Neutrófilos/patologia , Proteínas Proto-Oncogênicas/metabolismo , Sideróforos/metabolismo , Bexiga Urinária/patologia , Infecções Urinárias/imunologia , Infecções Urinárias/patologia , Adulto Jovem
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