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1.
Proc Natl Acad Sci U S A ; 116(8): 3100-3105, 2019 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-30718426

RESUMEN

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.


Asunto(s)
Toxina Diftérica/administración & dosificación , Interleucina-2/administración & dosificación , Melanoma Experimental/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/genética , Sustitución de Aminoácidos/genética , Anticuerpos/administración & dosificación , Proliferación Celular/efectos de los fármacos , Corynebacterium diphtheriae/química , Corynebacterium diphtheriae/patogenicidad , Toxina Diftérica/química , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Inmunosupresores/administración & dosificación , Inmunotoxinas/administración & dosificación , Interleucina-2/química , Subunidad alfa del Receptor de Interleucina-2/efectos de los fármacos , Melanoma Experimental/genética , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Linfocitos T Reguladores/efectos de los fármacos
2.
Biochemistry ; 57(1): 117-135, 2018 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-29039929

RESUMEN

Tabtoxinine-ß-lactam (TßL), also known as wildfire toxin, is a time- and ATP-dependent inhibitor of glutamine synthetase produced by plant pathogenic strains of Pseudomonas syringae. Here we demonstrate that recombinant glutamine synthetase from Escherichia coli phosphorylates the C3-hydroxyl group of the TßL 3-(S)-hydroxy-ß-lactam (3-HßL) warhead. Phosphorylation of TßL generates a stable, noncovalent enzyme-ADP-inhibitor complex that resembles the glutamine synthetase tetrahedral transition state. The TßL ß-lactam ring remains intact during enzyme inhibition, making TßL mechanistically distinct from traditional ß-lactam antibiotics such as penicillin. Our findings could enable the design of new 3-HßL transition state inhibitors targeting enzymes in the ATP-dependent carboxylate-amine ligase superfamily with broad therapeutic potential in many disease areas.


Asunto(s)
Adenosina Trifosfato/metabolismo , Azetidinas/farmacología , Toxinas Bacterianas/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Escherichia coli/enzimología , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Azetidinas/aislamiento & purificación , Azetidinas/metabolismo , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/aislamiento & purificación , Catálisis , Cromatografía Liquida , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Pseudomonas syringae/metabolismo
3.
J Invest Dermatol ; 142(4): 1126-1135.e4, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-34626614

RESUMEN

Signal transducer and activator of transcription 3 (STAT3) is important for psoriasis pathogenesis because STAT3 signaling downstream of IL-6, IL-21, IL-22, and IL-23 contributes to T helper type 17 cell development and because transgenic mice with keratinocyte (KC) STAT3 expression (K14-Stat3C mice) develop psoriasis-like dermatitis. In this study, the relative contribution of STAT3 signaling in KCs versus in T cells was evaluated in the imiquimod model of psoriasis-like dermatitis. Mice with STAT3-inducible deletion in KCs (K5-Stat3-/- mice) had decreased psoriasis-like dermatitis and epidermal STAT3 phosphorylation compared with wild-type mice, whereas mice with constitutive deletion of STAT3 in all T cells were similar to wild-type mice. Interestingly, mice with KC-inducible deletion of IL-6Rα had similar findings to those of K5-Stat3-/- mice, identifying IL-6/IL-6R as a predominant upstream signal for KC STAT3-induced psoriasis-like dermatitis. Moreover, psoriasis-like dermatitis inversely associated with type 1 immune gene products, especially CXCL10, whereas CXCL10 limited psoriasis-like dermatitis, suggesting that KC STAT3 signaling promoted psoriasis-like dermatitis by restricting downstream CXCL10 expression. Finally, treatment of mice with the pan-Jak inhibitor, tofacitinib, reduced psoriasis-like dermatitis and epidermal STAT3 phosphorylation. Taken together, STAT3 signaling in KCs rather than in T cells was a more important determinant for psoriasis-like dermatitis in a mechanism that involved upstream KC IL-6R signaling and downstream inhibition of type 1 immunity‒associated CXCL10 responses.


Asunto(s)
Dermatitis , Psoriasis , Animales , Quimiocina CXCL10 , Dermatitis/patología , Modelos Animales de Enfermedad , Interleucina-6/metabolismo , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Receptores de Interleucina-6 , Factor de Transcripción STAT3/metabolismo , Linfocitos T/metabolismo
4.
J Invest Dermatol ; 141(2): 274-284, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32943210

RESUMEN

Atopic dermatitis (AD) is a common, chronic, inflammatory skin condition characterized by recurrent and pruritic skin eruptions. Multiple factors contribute to the pathogenesis of AD, including skin barrier dysfunction, microbial dysbiosis, and immune dysregulation. Interactions among these factors form a complex, multidirectional network that can reinforce atopic skin disease but can also be ameliorated by targeted therapies. This review summarizes the complex interactions among contributing factors in AD and the implications on disease development and therapeutic interventions.


Asunto(s)
Dermatitis Atópica/etiología , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/inmunología , Disbiosis , Humanos , Microbiota , Piel/microbiología
5.
Sci Transl Med ; 13(601)2021 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-34233954

RESUMEN

Staphylococcus aureus causes most skin infections in humans, and the emergence of methicillin-resistant S. aureus (MRSA) strains is a serious public health threat. There is an urgent clinical need for nonantibiotic immunotherapies to treat MRSA infections and prevent the spread of antibiotic resistance. Here, we investigated the pan-caspase inhibitor quinoline-valine-aspartic acid-difluorophenoxymethyl ketone (Q-VD-OPH) for efficacy against MRSA skin infection in mice. A single systemic dose of Q-VD-OPH decreased skin lesion sizes and reduced bacterial burden compared with vehicle-treated or untreated mice. Although Q-VD-OPH inhibited inflammasome-dependent apoptosis-associated speck-like protein containing caspase activation and recruitment domain (ASC) speck formation and caspase-1-mediated interleukin-1ß (IL-1ß) production, Q-VD-OPH maintained efficacy in mice deficient in IL-1ß, ASC, caspase-1, caspase-11, or gasdermin D. Thus, Q-VD-OPH efficacy was independent of inflammasome-mediated pyroptosis. Rather, Q-VD-OPH reduced apoptosis of monocytes and neutrophils. Moreover, Q-VD-OPH enhanced necroptosis of macrophages with concomitant increases in serum TNF and TNF-producing neutrophils, monocytes/macrophages, and neutrophils in the infected skin. Consistent with this, Q-VD-OPH lacked efficacy in mice deficient in TNF (with associated reduced neutrophil influx and necroptosis), in mice deficient in TNF/IL-1R and anti-TNF antibody-treated WT mice. In vitro studies revealed that combined caspase-3, caspase-8, and caspase-9 inhibition reduced apoptosis, and combined caspase-1, caspase-8, and caspase-11 inhibition increased TNF, suggesting a mechanism for Q-VD-OPH efficacy in vivo. Last, Q-VD-OPH also had a therapeutic effect against Streptococcus pyogenes and Pseudomonas aeruginosa skin infections in mice. Collectively, pan-caspase inhibition represents a potential host-directed immunotherapy against MRSA and other bacterial skin infections.


Asunto(s)
Caspasas , Staphylococcus aureus Resistente a Meticilina , Animales , Caspasa 1 , Inhibidores de Caspasas/farmacología , Inmunoterapia , Inflamasomas , Interleucina-1beta , Ratones , Inhibidores del Factor de Necrosis Tumoral
6.
J Clin Invest ; 131(5)2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33645549

RESUMEN

IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL­36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL­36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL­36 cytokines in human atopic dermatitis skin and in IL­36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL­36 responses represent a key mechanism and potential therapeutic target against allergic diseases.


Asunto(s)
Dermatitis Atópica/inmunología , Inmunoglobulina E/inmunología , Interleucina-1/inmunología , Queratinocitos/inmunología , Células Plasmáticas/inmunología , Staphylococcus aureus/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Dermatitis Atópica/genética , Dermatitis Atópica/microbiología , Humanos , Cambio de Clase de Inmunoglobulina , Inmunoglobulina E/genética , Interleucina-1/genética , Interleucina-4/genética , Interleucina-4/inmunología , Queratinocitos/microbiología , Ratones , Ratones Noqueados , Células Plasmáticas/patología
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