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The aim of this case report is to review and compare the clinical, radiologic, histopathologic, and immunohistochemical features, along with the treatment of a case of ghost cell odontogenic carcinoma. In addition, a report of the existing published literature with an emphasis on treatment will be described to provide information on this rare but aggressive tumor. The family of odontogenic ghost cell tumors comprises a spectrum of lesions characterized by odontogenic epithelium with ghost cell keratinization and calcifications. It appears that early detection is vital in proper treatment due to the high possibility of malignant transformation.
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Carcinoma , Neoplasias Maxilomandibulares , Tumores Odontogênicos , Humanos , Tumores Odontogênicos/diagnóstico por imagem , Tumores Odontogênicos/cirurgia , Neoplasias Maxilomandibulares/patologia , Epitélio , Transformação Celular NeoplásicaRESUMO
Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.
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Doenças Autoimunes , Dependovirus , Ratos , Camundongos , Animais , Peptídeos/farmacologia , Doenças Autoimunes/tratamento farmacológico , Obesidade , FígadoRESUMO
Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.
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Artrite Reumatoide , Probióticos , Ratos , Humanos , Animais , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Peptídeos/metabolismo , Artrite Reumatoide/tratamento farmacológico , Inflamação/tratamento farmacológico , Probióticos/uso terapêutico , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/uso terapêuticoRESUMO
The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.
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Canal de Sódio Disparado por Voltagem NAV1.7 , Dor , Peptídeos , Bloqueadores do Canal de Sódio Disparado por Voltagem , Animais , Humanos , Camundongos , Ratos , Nociceptores , Dor/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Desenho de FármacosRESUMO
Natural peptides isolated from animal venoms generally target cell surface receptors with high affinity and selectivity. On many occasions, some of these receptors are over-expressed in cancer cells. Herein, we identified Lqh-8/6 as a natural peptide analog of chlorotoxin, a proven and useful compound for the diagnosis and treatment of glioma. Lqh-8/6 and two other natural analogues were chemically synthesized for the first time and evaluated for their ability to label, detect and prevent glioma growth in vitro. We demonstrate that a biotinylated version of Lqh-8/6 allows both the labeling of glioma cell lines and the detection of glioma in brain sections of glioma allograft Fisher rats. Lqh-8/6 has intrinsic anti-invasive properties but is non-toxic to glioma cells. To confer anti-tumor properties to Lqh-8/6, we chemically coupled doxorubicin to the glioma-targeting peptide using click chemistry. To this end, we successfully chemically synthesized Lqh-8/6-azide and doxorubicin-alkyne without impairing the toxic nature of doxorubicin. The toxin-drug conjugate efficiently promotes the apoptosis of glioma cells in vitro. This example contributes to the concept that animal venom peptides constitute exquisite warheads for delivering toxic chemical conjugates, a parallel to the popular concept of antibody-drug conjugates for the treatment of cancer.
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Voltage-gated Kv1.3 potassium channels are key regulators of T lymphocyte activation, proliferation and cytokine production, by providing the necessary membrane hyper-polarization for calcium influx following immune stimulation. It is noteworthy that an accumulating body of in vivo and in vitro evidence links these channels to multiple sclerosis pathophysiology. Here we studied the electrophysiological properties and the transcriptional and translational expression of T lymphocyte Kv1.3 channels in multiple sclerosis, by combining patch clamp recordings, reverse transcription polymerase chain reaction and flow cytometry on freshly isolated peripheral blood T lymphocytes from two patient cohorts with multiple sclerosis, as well as from healthy and disease controls. Our data demonstrate that T lymphocytes in MS, manifest a significant up-regulation of Kv1.3 mRNA, Kv1.3 membrane protein and Kv1.3 current density and therefore of functional membrane channel protein, compared to control groups (p < 0.001). Interestingly, patient sub-grouping shows that Kv1.3 channel density is significantly higher in secondary progressive, compared to relapsing-remitting multiple sclerosis (p < 0.001). Taking into account the tight connection between Kv1.3 channel activity and calcium-dependent processes, our data predict and could partly explain the reported alterations of T lymphocyte function in multiple sclerosis, while they highlight Kv1.3 channels as potential therapeutic targets and peripheral biomarkers for the disease.
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BACKGROUND AND PURPOSE: KV 1.3 channels are expressed in vascular smooth muscle cells (VSMCs), where they contribute to proliferation rather than contraction and participate in vascular remodelling. KV 1.3 channels are also expressed in macrophages, where they assemble with KV 1.5 channels (KV 1.3/KV 1.5), whose activation generates a KV current. In macrophages, the KV 1.3/KV 1.5 ratio is increased by classical activation (M1). Whether these channels are involved in angiotensin II (AngII)-induced vascular remodelling, and whether they can modulate the macrophage phenotype in hypertension, remains unknown. We characterized the role of KV 1.3 channels in vascular damage in hypertension. EXPERIMENTAL APPROACH: We used AngII-infused mice treated with two selective KV 1.3 channel inhibitors (HsTX[R14A] and [EWSS]ShK). Vascular function and structure were measured using wire and pressure myography, respectively. VSMC and macrophage electrophysiology were studied using the patch-clamp technique; gene expression was analysed using RT-PCR. KEY RESULTS: AngII increased KV 1.3 channel expression in mice aorta and peritoneal macrophages which was abolished by HsTX[R14A] treatment. KV 1.3 inhibition did not prevent hypertension, vascular remodelling, or stiffness but corrected AngII-induced macrophage infiltration and endothelial dysfunction in the small mesenteric arteries and/or aorta, via a mechanism independent of electrophysiological changes in VSMCs. AngII modified the electrophysiological properties of peritoneal macrophages, indicating an M1-like activated state, with enhanced expression of proinflammatory cytokines that induced endothelial dysfunction. These effects were prevented by KV 1.3 blockade. CONCLUSIONS AND IMPLICATIONS: We unravelled a new role for KV 1.3 channels in the macrophage-dependent endothelial dysfunction induced by AngII in mice which might be due to modulation of macrophage phenotype.
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Angiotensina II , Hipertensão , Angiotensina II/toxicidade , Animais , Hipertensão/induzido quimicamente , Macrófagos , Camundongos , Miócitos de Músculo Liso , Remodelação VascularRESUMO
We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.
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Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.
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Potenciais da Membrana , Trifosfato de Adenosina , Doença de Alzheimer , Animais , Cálcio , Feminino , Inflamação , Microglia , Receptores Purinérgicos P2 , Receptores Purinérgicos P2X4RESUMO
Pain is a significant public health burden in the United States, and current treatment approaches rely heavily on opioids, which often have limited efficacy and can lead to addiction. In humans, functional loss of the voltage-gated sodium channel Nav1.7 leads to pain insensitivity without deficits in the central nervous system. Accordingly, discovery of a selective Nav1.7 antagonist should provide an analgesic without abuse liability and an improved side-effect profile. Huwentoxin-IV, a component of tarantula venom, potently blocks sodium channels and is an attractive scaffold for engineering a Nav1.7-selective molecule. To define the functional impact of alterations in huwentoxin-IV sequence, we produced a library of 373 point mutants and tested them for Nav1.7 and Nav1.2 activity. We then combined favorable individual changes to produce combinatorial mutants that showed further improvements in Nav1.7 potency (E1N, E4D, Y33W, Q34S-Nav1.7 pIC50 = 8.1 ± 0.08) and increased selectivity over other Nav isoforms (E1N, R26K, Q34S, G36I, Nav1.7 pIC50 = 7.2 ± 0.1, Nav1.2 pIC50 = 6.1 ± 0.18, Nav1.3 pIC50 = 6.4 ± 1.0), Nav1.4 is inactive at 3 µm, and Nav1.5 is inactive at 10 µm We also substituted noncoded amino acids at select positions in huwentoxin-IV. Based on these results, we identify key determinants of huwentoxin's Nav1.7 inhibition and propose a model for huwentoxin-IV's interaction with Nav1.7. These findings uncover fundamental features of huwentoxin involved in Nav1.7 blockade, provide a foundation for additional optimization of this molecule, and offer a basis for the development of a safe and effective analgesic.
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Analgésicos/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Venenos de Aranha/química , Venenos de Aranha/genética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Sequência de Aminoácidos/genética , Desenvolvimento de Medicamentos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Mutagênese , Canal de Sódio Disparado por Voltagem NAV1.2/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.2/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/tratamento farmacológico , Biblioteca de Peptídeos , Mutação Puntual , Engenharia de Proteínas , Isoformas de Proteínas , Proteínas Recombinantes , Venenos de Aranha/isolamento & purificaçãoRESUMO
The global spread of multidrug-resistant strains of Neisseria gonorrhoeae constitutes a public health emergency. With limited antibiotic treatment options, there is an urgent need for development of a safe and effective vaccine against gonorrhea. Previously, we constructed a prototype vaccine candidate comprising a peptide mimic (mimitope) of a glycan epitope on gonococcal lipooligosaccharide (LOS), recognized by monoclonal antibody 2C7. The 2C7 epitope is (i) broadly expressed as a gonococcal antigenic target in human infection, (ii) a critical requirement for gonococcal colonization in the experimental setting, and (iii) a virulence determinant that is maintained and expressed by gonococci. Here, we have synthesized to >95% purity through a relatively facile and economical process a tetrapeptide derivative of the mimitope that was cyclized through a nonreducible thioether bond, thereby rendering the compound homogeneous and stable. This vaccine candidate, called TMCP2, when administered at 0, 3, and 6 weeks to BALB/c mice at either 50, 100 or 200 µg/dose in combination with glucopyranosyl lipid A-stable oil-in-water nanoemulsion (GLA-SE; a Toll-like receptor 4 and TH1-promoting adjuvant), elicited bactericidal IgG and reduced colonization levels of gonococci in experimentally infected mice while accelerating clearance by each of two different gonococcal strains. Similarly, a 3-dose biweekly schedule (50 µg TMCP2/dose) was also effective in mice. We have developed a gonococcal vaccine candidate that can be scaled up and produced economically to a high degree of purity. The candidate elicits bactericidal antibodies and is efficacious in a preclinical experimental infection model.IMPORTANCENeisseria gonorrhoeae has become resistant to most antibiotics. The incidence of gonorrhea is also sharply increasing. A safe and effective antigonococcal vaccine is urgently needed. Lipooligosaccharide (LOS), the most abundant outer membrane molecule, is indispensable for gonococcal pathogenesis. A glycan epitope on LOS that is recognized by monoclonal antibody (MAb) 2C7 (called the 2C7 epitope) is expressed almost universally by gonococci in vivo Previously, we identified a peptide mimic (mimitope) of the 2C7 epitope, which when configured as an octamer and used as an immunogen, attenuated colonization of mice by gonococci. Here, a homogenous, stable tetrameric derivative of the mimitope, when combined with a TH1-promoting adjuvant and used as an immunogen, also effectively attenuates gonococcal colonization of mice. This candidate peptide vaccine can be produced economically, an important consideration for gonorrhea, which affects socioeconomically underprivileged populations disproportionately, and represents an important advance in the development of a gonorrhea vaccine.
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Vacinas Bacterianas/imunologia , Lipopolissacarídeos/imunologia , Neisseria gonorrhoeae/imunologia , Peptídeos/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Epitopos/imunologia , Feminino , Gonorreia/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Dinosaur fossils possessing integumentary appendages of various morphologies, interpreted as feathers, have greatly enhanced our understanding of the evolutionary link between birds and dinosaurs, as well as the origins of feathers and avian flight. In extant birds, the unique expression and amino acid composition of proteins in mature feathers have been shown to determine their biomechanical properties, such as hardness, resilience, and plasticity. Here, we provide molecular and ultrastructural evidence that the pennaceous feathers of the Jurassic nonavian dinosaur Anchiornis were composed of both feather ß-keratins and α-keratins. This is significant, because mature feathers in extant birds are dominated by ß-keratins, particularly in the barbs and barbules forming the vane. We confirm here that feathers were modified at both molecular and morphological levels to obtain the biomechanical properties for flight during the dinosaur-bird transition, and we show that the patterns and timing of adaptive change at the molecular level can be directly addressed in exceptionally preserved fossils in deep time.
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Evolução Molecular , Plumas/química , Queratinas/química , beta-Queratinas/química , Animais , Aves , Dinossauros , Plumas/ultraestrutura , Fósseis , Pele/química , Pele/ultraestruturaRESUMO
BACKGROUND: Fibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA. METHODS: We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies. RESULTS: We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA. CONCLUSIONS: Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.
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Artrite Experimental/metabolismo , Fibroblastos/metabolismo , Canal de Potássio Kv1.3/fisiologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Sinoviócitos/metabolismo , Linfócitos T/metabolismo , Animais , Artrite Experimental/diagnóstico por imagem , Artrite Reumatoide/diagnóstico por imagem , Artrite Reumatoide/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Ratos , Ratos Endogâmicos LewRESUMO
BACKGROUND: Disease-associated-microglia (DAM) represent transcriptionally-distinct and neurodegeneration-specific microglial profiles with unclear significance in Alzheimer's disease (AD). An understanding of heterogeneity within DAM and their key regulators may guide pre-clinical experimentation and drug discovery. METHODS: Weighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to identify modules of highly co-expressed genes. These modules were contrasted with known signatures of homeostatic microglia and DAM to reveal novel molecular heterogeneity within DAM. Flow cytometric validation studies were performed to confirm existence of distinct DAM sub-populations in AD mouse models predicted by WGCNA. Gene ontology analyses coupled with bioinformatics approaches revealed drug targets and transcriptional regulators of microglial modules predicted to favorably modulate neuroinflammation in AD. These guided in-vivo and in-vitro studies in mouse models of neuroinflammation and neurodegeneration (5xFAD) to determine whether inhibition of pro-inflammatory gene expression and promotion of amyloid clearance was feasible. We determined the human relevance of these findings by integrating our results with AD genome-wide association studies and human AD and non-disease post-mortem brain proteomes. RESULTS: WGCNA applied to microglial gene expression data revealed a transcriptomic framework of microglial activation that predicted distinct pro-inflammatory and anti-inflammatory phenotypes within DAM, which we confirmed in AD and aging models by flow cytometry. Pro-inflammatory DAM emerged earlier in mouse models of AD and were characterized by pro-inflammatory genes (Tlr2, Ptgs2, Il12b, Il1b), surface marker CD44, potassium channel Kv1.3 and regulators (NFkb, Stat1, RelA) while anti-inflammatory DAM expressed phagocytic genes (Igf1, Apoe, Myo1e), surface marker CXCR4 with distinct regulators (LXRα/ß, Atf1). As neuro-immunomodulatory strategies, we validated LXRα/ß agonism and Kv1.3 blockade by ShK-223 peptide that promoted anti-inflammatory DAM, inhibited pro-inflammatory DAM and augmented Aß clearance in AD models. Human AD-risk genes were highly represented within homeostatic microglia suggesting causal roles for early microglial dysregulation in AD. Pro-inflammatory DAM proteins were positively associated with neuropathology and preceded cognitive decline confirming the therapeutic relevance of inhibiting pro-inflammatory DAM in AD. CONCLUSIONS: We provide a predictive transcriptomic framework of microglial activation in neurodegeneration that can guide pre-clinical studies to characterize and therapeutically modulate neuroinflammation in AD.
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Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Microglia/metabolismo , Microglia/patologia , Animais , Estudo de Associação Genômica Ampla , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , TranscriptomaRESUMO
Sea anemone venom is rich in bioactive compounds, including peptides containing multiple disulfide bridges. In a transcriptomic study on Oulactis sp., we identified the putative 36-residue peptide, OspTx2b, which is an isoform of the KV channel blocker OspTx2a (Sunanda P et al. [2018] Identification, chemical synthesis, structure and function of a new KV1 channel blocking peptide from Oulactis sp. Peptide Science, in press). As OspTx2b contains a ShK/BgK-like cysteine framework, with high amino acid sequence similarity to BgK, we were interested to investigate its structure and function. The solution structure of OspTx2b was determined using nuclear magnetic resonance spectroscopy. OspTx2b does indeed possess a BgK-like scaffold, with the same disulfide bond connectivities. The orientation of the Lys-Tyr dyad in OspTx2b is more similar to that in ShK than in BgK. However, it failed to show against a range of voltage-gated potassium channels in Xenopus oocytes and human T lymphocytes. OspTx2b also showed no growth inhibitory activity against several strains of bacteria and fungi. Having a BgK-like fold with the Lys-Tyr dyad but no BgK-like activity highlights the importance of key amino acid residues in BgK that are missing in OspTx2b. The lack of activity against the KV channels assessed in this study emphasises that the ShK/BgK scaffold is capable of supporting functional activity beyond potassium channel blockade.
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Venenos de Cnidários/química , Venenos de Cnidários/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Anêmonas-do-Mar , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Modelos Moleculares , Oócitos , Conformação Proteica , Dobramento de Proteína , Xenopus laevisRESUMO
Fibroblast-like synoviocytes (FLSs) are a key cell type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLSs and found that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects. In this study, we aimed to determine whether the KCa1.1 ß1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch-clamp and functional assays were used to determine whether IbTX can regulate FLSs through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined whether IbTX causes side effects including incontinence or tremors in rats, compared with those treated with the small-molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 were expressed by FLSs in joints of rats with experimental arthritis. IbTX inhibited KCa1.1 channels expressed by FLSs from patients with RA and by FLSs from rat models of RA and reduced FLS invasiveness. IbTX significantly reduced disease severity in two rat models of RA. Unlike paxilline, IbTX did not induce tremors or incontinence in rats. Overall, IbTX inhibited KCa1.1 channels on FLSs and treated rat models of RA without inducing side effects associated with nonspecific KCa1.1 blockade and could become the basis for the development of a new treatment of RA.
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Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Terapia de Alvo Molecular , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/química , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Peptídeos/uso terapêutico , Bloqueadores dos Canais de Potássio/uso terapêutico , Ratos , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/metabolismoRESUMO
Peptide toxins elaborated by sea anemones target various ion-channel sub-types. Recent transcriptomic studies of sea anemones have identified several novel candidate peptides, some of which have cysteine frameworks identical to those of previously reported sequences. One such peptide is AsK132958, which was identified in a transcriptomic study of Anemonia sulcata and has a cysteine framework similar to that of ShK from Stichodactyla helianthus, but is six amino acid residues shorter. We have determined the solution structure of this novel peptide using NMR spectroscopy. The disulfide connectivities and structural scaffold of AsK132958 are very similar to those of ShK but the structure is more constrained. Toxicity assays were performed using grass shrimp (Palaemonetes sp) and Artemia nauplii, and patch-clamp electrophysiology assays were performed to assess the activity of AsK132958 against a range of voltage-gated potassium (KV) channels. AsK132958 showed no activity against grass shrimp, Artemia nauplii, or any of the KV channels tested, owing partly to the absence of a functional Lys-Tyr dyad. Three AsK132958 analogues, each containing a Tyr in the vicinity of Lys19, were therefore generated in an effort to restore binding, but none showed activity against any of KV channels tested. However, AsK132958 and its analogues are less susceptible to proteolysis than that of ShK. Our structure suggests that Lys19, which might be expected to occupy the pore of the channel, is not sufficiently accessible for binding, and therefore that AsK132958 must have a distinct functional role that does not involve KV channels.
Assuntos
Venenos de Cnidários/química , Peptídeos/química , Bloqueadores dos Canais de Potássio/química , Dobramento de Proteína , Anêmonas-do-Mar/química , Animais , Venenos de Cnidários/farmacologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Xenopus laevisRESUMO
Peptides are recognized as being highly selective, potent and relatively safe as potential therapeutics. Peptides isolated from the venom of different animals satisfy most of these criteria with the possible exception of safety, but when isolated as single compounds and used at appropriate concentrations, venom-derived peptides can become useful drugs. Although the number of venom-derived peptides that have successfully progressed to the clinic is currently limited, the prospects for venom-derived peptides look very optimistic. As proteomic and transcriptomic approaches continue to identify new sequences, the potential of venom-derived peptides to find applications as therapeutics, cosmetics and insecticides grows accordingly.
Assuntos
Descoberta de Drogas/métodos , Peptídeos/química , Peptídeos/uso terapêutico , Peçonhas/química , Peçonhas/uso terapêutico , Sequência de Aminoácidos , Animais , Ensaios Clínicos como Assunto , Cosméticos/química , Aprovação de Drogas , Humanos , Inseticidas/química , Modelos Moleculares , Peptídeos/farmacologia , Proteômica/métodos , Peçonhas/farmacologiaRESUMO
The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.