Assuntos
Artrite Psoriásica , Dermatite Atópica , Doenças Inflamatórias Intestinais , Psoríase , Células Th17 , Células Th2 , Humanos , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/complicações , Células Th17/imunologia , Psoríase/tratamento farmacológico , Psoríase/complicações , Psoríase/imunologia , Células Th2/imunologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/complicações , Artrite Psoriásica/tratamento farmacológico , Artrite Psoriásica/complicações , Masculino , Feminino , Adulto , Quimioterapia Combinada , Pessoa de Meia-Idade , Terapia Biológica/métodos , Resultado do TratamentoRESUMO
Post-hyaluronic acid filler nodules are uncommon, unpredictable complications that present a challenge to clinical therapy. We report a case of a female in her fifties who developed edema and nodules 6 weeks after hyaluronic acid (HA) filler injection. After minimal improvement with oral steroids and intralesional hyaluronidase, a trial of oral abrocitinib was initiated, which yielded significant clinical improvement. Thus, abrocitinib may be a novel therapeutic option for delayed-onset nodules following injection of hyaluronic acid. J Drugs Dermatol. 2024;23(1):1355-1356. doi:10.36849/JDD.7271.
Assuntos
Excipientes , Ácido Hialurônico , Pirimidinas , Humanos , Feminino , Ácido Hialurônico/efeitos adversos , Hialuronoglucosaminidase , SulfonamidasRESUMO
Kresch M, Guénin S, Mubasher A, et al. Talquetamab-induced Grover’s disease. J Drugs Dermatol. 2023;22(8):828-829. doi:10.36849/JDD.7170.
Assuntos
Acantólise , Ictiose , Humanos , Acantólise/diagnóstico , Acantólise/etiologiaRESUMO
Guénin S, Kazemi A, Cline A, et al. Rethinking the inflammatory balance in psoriasis and atherosclerosis. J Drugs Dermatol. 2023;22(8):832-833. doi:10.36849/JDD.7082.
Assuntos
Aterosclerose , Psoríase , Humanos , Psoríase/complicações , Psoríase/diagnóstico , Aterosclerose/diagnóstico , Aterosclerose/etiologia , Resultado do TratamentoRESUMO
D3/D2 sub-specificity is a complex problem to solve. Indeed, in the absence of easy structural biology of the G-protein coupled receptors, and despite key progresses in this area, the systematic knowledge of the ligand/receptor relationship is difficult to obtain. Due to these structural biology limitations concerning membrane proteins, we favored the use of directed mutagenesis to document a rational towards the discovery of markedly specific D3 ligands over D2 ligands together with basic binding experiments. Using our methodology of stable expression of receptors in HEK cells, we constructed the gene encoding for 24 mutants and 4 chimeras of either D2 or D3 receptors and expressed them stably. Those cell lines, expressing a single copy of one receptor mutant each, were stably constructed, selected, amplified and the membranes from them were prepared. Binding data at those receptors were obtained using standard binding conditions for D2 and D3 dopamine receptors. We generated 26 new molecules derived from D2 or D3 ligands. Using 8 reference compounds and those 26 molecules, we characterized their binding at those mutants and chimeras, exemplifying an approach to better understand the difference at the molecular level of the D2 and D3 receptors. Although all the individual results are presented and could be used for minute analyses, the present report does not discuss the differences between D2 and D3 data. It simply shows the feasibility of the approach and its potential.
Assuntos
Receptores de Dopamina D2 , Receptores de Dopamina D3 , Receptores de Dopamina D3/genética , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Ligantes , Linhagem Celular , MutagêneseRESUMO
Cloning may seem to be a view from the past. The time before software, computers and AI were invented. It seems to us worth discussing these points in view of our favorite target: the melatoninergic system. In a few stances, it might be important to point out that even in the new era of dry science, there is still a need to experiment and to prove at the bench that our in silico assertions are right. Most of the living animals express to some extend the melatonin receptors. Some of these animal genomes were completely or partially sequenced, and it is tempting to extract from this huge information the sequence(s) of our favorite genes (MLT receptors). Then, why bother cloning, as opposed to simply built the gene and express it in a host cell? Because the genetic boundaries of the expressed sequence(s) are not 100% sure. Because the melatonin receptor gene(s) comprise a first exon 25,000 base pair far from the second one and the limits between this Ex1 and In1-as between In1 and Ex2-are subject to changes that might have a huge impact on the biochemical properties of the receptor, once expressed. Because a receptor is a biochemical entity with characteristics that are important for the functioning of this particular pathway, and more generally, for the functioning of life.
Assuntos
Melatonina , Animais , Clonagem Molecular , Éxons , Melatonina/metabolismo , Receptores de Melatonina/genéticaRESUMO
VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2 -Fc-VHH2 . The structure of VHH2 -Fc-VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2 -Fc-VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2 -Fc). This new type of molecules, VHH2 -Fc-VHH2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.
Assuntos
Complexo Antígeno-Anticorpo/química , Antígenos/química , Fragmentos Fc das Imunoglobulinas/química , Receptor ErbB-2/química , Proteínas Recombinantes de Fusão/química , Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/metabolismo , Antígenos/genética , Antígenos/metabolismo , Camelus , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Peso Molecular , Ligação Proteica , Engenharia de Proteínas/métodos , Multimerização Proteica , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/metabolismoRESUMO
Hibernation is an exceptional physiological response to a hostile environment, characterized by a seasonal period of torpor cycles involving dramatic reductions of body temperature and metabolism, and arousal back to normothermia. As the mechanisms regulating hibernation are still poorly understood, here we analysed the expression of genes involved in energy homeostasis, torpor regulation, and daily or seasonal timing using digital droplet PCR in various central and peripheral tissues sampled at different stages of torpor/arousal cycles in the European hamster. During torpor, the hypothalamus exhibited strongly down-regulated gene expression, suggesting that hypothalamic functions were reduced during this period of low metabolic activity. During both torpor and arousal, many structures (notably the brown adipose tissue) exhibited altered expression of deiodinases, potentially leading to reduced tissular triiodothyronine availability. During the arousal phase, all analysed tissues showed increased expression of the core clock genes Per1 and Per2. Overall, our data indicated that the hypothalamus and brown adipose tissue were the tissues most affected during the torpor/arousal cycle, and that clock genes may play critical roles in resetting the body's clocks at the beginning of the active period.
Assuntos
Tecido Adiposo Marrom/metabolismo , Nível de Alerta/genética , Cricetulus/genética , Metabolismo Energético/genética , Hibernação/genética , Hipotálamo/metabolismo , Proteínas Circadianas Period/genética , Animais , Ritmo Circadiano/genética , Cricetulus/metabolismo , Europa (Continente) , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Masculino , Anotação de Sequência Molecular , Proteínas Circadianas Period/metabolismo , Tri-Iodotironina/metabolismoRESUMO
For many years, it was of interest to identify the sequences encoding the two melatonin receptors (MT1 and MT2) from various species. After publishing the basic molecular characterization of the human, rat, mouse, sheep, and platypus MT1, MT2, or Mel1c receptors, we began cloning the genes from other animals, such as birds, bats, and vipers. The goal was to advance the receptor crystallization, which could greatly contribute the understanding of the sequence/stability relationship. European hamster MT1 receptor was cloned for the first time from this gender, was expressed in stable form in cells, and its binding characterized with a sample of 19 melatonin ligands. Siberian hamster (Phodopus sungorus) expresses a non-functional MT2. We observed that unlike this hamster, the European hamster (Cricetus cricetus) does not have a stop codon in the MT2 sequence. Thus, we undertook the tedious task of cloning the MT2 receptor. We partially succeeded, sequencing the complete exon 2 and a fragment of exon 1 (from putative amino acids 12 to 38 and 77 to 323), after several years of efforts. In order to show that the protein parts we cloned were capable to sustain some binding capacities, we designed a chimeric MT2 receptor using a consensus sequence to replace the unknown amino acids, based on other small rodent MT2 sequences. This chimeric construct could bind melatonin in the nanomolar range. This work is meant to be the basis for attempts from other laboratories of the community to determine the complete natural sequence of the European hamster MT2 receptor. The present work is the first to show that, among the hamsters, if the Siberian is a natural knockout for MT2, the European one is not.
Assuntos
Cricetinae/genética , Melatonina/metabolismo , Receptor MT1 de Melatonina/genética , Receptor MT2 de Melatonina/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Códon de Terminação , Éxons , Ligantes , Masculino , Ligação Proteica , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals.
Assuntos
Melatonina/metabolismo , Ornitorrinco/metabolismo , Receptores de Melatonina/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Clonagem Molecular/métodos , Filogenia , Ornitorrinco/genética , Ligação Proteica , Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/química , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Receptores de Melatonina/química , Receptores de Melatonina/genéticaRESUMO
Refractory immune thrombocytopenia (ITP) was previously defined as lack of a minimum response to splenectomy and the requirement for long-term treatment to reduce the risk of significant bleeding events. In this multicenter study, we included 37 patients with multirefractory ITP, defined as no response to splenectomy, rituximab, romiplostim, and eltrombopag. As compared with a historical cohort of 183 ITP patients, matched on the calendar year of ITP diagnosis with a 5:1 ratio, patients with multirefractory ITP were more likely to have secondary ITP (odds ratio [OR], 4.84; 95% confidence interval [CI], 1.31-17.86; P = .018) and monoclonal gammopathy of undetermined significance (OR, 5.94; 95% CI, 1.08-32.48; P = .04). The median duration of ITP before being recognized as multirefractory was 78 months (range, 6-450). The patients showed failure of a median of 10.5 prior treatment lines for ITP (range, 6-15). At the end of follow-up (median, 84 months; range, 12-455), only 1/14 patients achieved response with immunosuppressant therapy alone. By contrast, 7/10 patients achieved response with a combination of immunosuppressant therapy and thrombopoietin-receptor agonists that lasted for a median of 15 months (range, 6-32). Throughout the course of ITP, 5/37 patients died, 3 with ITP (bleeding, n = 2; sepsis n = 1); 15 (40%) had at least 1 bacterial infection and 9 (24%) at least 1 episode of thrombosis. In conclusion, multirefractory ITP was associated with high morbidity and mortality. Combining an immunosuppressant therapy with thrombopoietin-receptor agonists may be a good strategy for management for these patients with severe disease.
Assuntos
Antineoplásicos/uso terapêutico , Resistência a Medicamentos , Púrpura Trombocitopênica Idiopática/terapia , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Rituximab/uso terapêutico , Esplenectomia/efeitos adversos , Trombopoetina/uso terapêutico , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Doença Crônica , Terapia Combinada , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²47) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Dissulfetos/metabolismo , Tiorredoxinas/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Clonagem Molecular , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Humanos , Oxirredução , Redobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.
Assuntos
Receptor MT1 de Melatonina/química , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/química , Receptor MT2 de Melatonina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Genômica , Humanos , Camundongos , Dados de Sequência Molecular , Transporte Proteico , Ratos , Alinhamento de Sequência , Ovinos , Especificidade da EspécieRESUMO
The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS.