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AIM: To characterize vestibular recovery in a mouse model of unilateral labyrinthotomy under local AAT and dexamethasone treatment. BACKGROUND: Alpha1-antitrypsin (AAT) is a circulating tissue-protective molecule that rises during inflammatory conditions and promotes inflammatory resolution. Its local concentration in human perilymph inversely correlates with the severity of inner ear dysfunction; concomitantly, mice that overexpress AAT and undergo inner ear trauma rapidly restore vestibular function. Locally applied AAT has yet to be examined in this context, nor has it been directly compared with anti-inflammatory corticosteroid treatment. METHODS: Wild-type mice C57BL/6 underwent a unilateral inner ear injury. Nine microliters of saline, clinical-grade AAT (180 µg/site), dexamethasone (4 mg/site), or both were applied locally on Days 0, 1, and 2 (n = 5/group). Vestibular function was assessed for 7 days. An in vitro human epithelial gap closure assay was performed using A549 cells in the presence of AAT and/or dexamethasone. RESULTS: Upon labyrinthotomy, all groups displayed severe vestibular dysfunction. Saline-treated mice showed the longest impairment. That group and the dexamethasone group displayed partial to no recovery, while AAT-treated mice exhibited complete recovery within 7 days; at this time point, dexamethasone-treated mice exhibited 50% recovery. Objective vestibular testing showed similar outcomes. In vitro, cotreatment with AAT and dexamethasone resulted in a gap closure dynamic that was superior to AAT alone at 6 h and superior to DEX alone at 48 h. CONCLUSION: Locally applied AAT treatment is superior to locally applied dexamethasone in promoting vestibular recovery in vivo. Ongoing studies are exploring the potential advantages of AAT combined with early low-dose dexamethasone therapy.
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Dexametasona , Ratones Endogámicos C57BL , alfa 1-Antitripsina , Animales , Ratones , Dexametasona/administración & dosificación , Dexametasona/uso terapéutico , alfa 1-Antitripsina/administración & dosificación , Enfermedades Vestibulares/tratamiento farmacológico , Enfermedades Vestibulares/etiología , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Oído Interno/efectos de los fármacos , Modelos Animales de Enfermedad , Recuperación de la Función/efectos de los fármacos , Vestíbulo del Laberinto/efectos de los fármacos , Vestíbulo del Laberinto/lesiones , Humanos , MasculinoRESUMEN
BACKGROUND: Most tympanic membrane (TM) perforations heal spontaneously, but 10%-20% remain chronic and might lead to impaired hearing and recurrent middle ear infections. Alpha1-antitrypsin (AAT) is a circulating tissue-protective protein that is elevated under inflammatory conditions and is currently indicated for genetic AAT deficiency. Recently, AAT has been shown to promote tissue remodeling and inflammatory resolution. OBJECTIVE: This study aimed to examine the effects of local clinical-grade AAT treatment on tissue repair in a mouse model of acute traumatic TM perforation. METHODS: Wild-type mice underwent unilateral TM perforation and were either left untreated or treated locally with human AAT (9 × 10-3 mL at 20 mg/mL on days 0, 1, and 2; n = 15/group). The perforations were evaluated macroscopically on a serial basis. Mice were sacrificed on various days post-injury, and TMs were excised for gene analysis by RT-PCR. RESULTS: There were no adverse reactions in hAAT-treated ears throughout the study period. Compared with untreated animals, TM closure occurred earlier in the treated group (days until full closure, median: 4 and 9, respectively). According to gene expression analysis, VEGF, TGFß, and collagen-5A1 were induced earlier in AAT-treated mice (day 4-5 compared with day 9). Additionally, IL-10 expression levels were higher and IL-6 levels were lower in treated versus untreated mice. CONCLUSION: A local tissue environment rich in AAT promotes early tissue repair in a perforated TM model both macroscopically and molecularly. Studies are underway to examine TM functionality and recombinant AAT formulations for micro-dosing in the format of a single local application. LEVEL OF EVIDENCE: NA Laryngoscope, 134:3802-3806, 2024.
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Modelos Animales de Enfermedad , Perforación de la Membrana Timpánica , Cicatrización de Heridas , alfa 1-Antitripsina , Animales , Ratones , Perforación de la Membrana Timpánica/tratamiento farmacológico , alfa 1-Antitripsina/farmacología , Cicatrización de Heridas/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
α1-Antitrypsin (AAT), an acute-phase reactant not unsimilar to C-reactive protein (CRP), is a serine protease inhibitor that harbors tissue-protective and immunomodulatory attributes. Its concentrations appropriately increase during conditions of extensive tissue injury, and it induces immune tolerance, in part, by inhibiting the enzymatic activity of the inflammatory serine protease, proteinase 3 (PR3). Typically administered to patients with genetic AAT deficiency, AAT treatment was recently shown to improve outcomes in patients with steroid-refractory graft-versus-host disease (GVHD). GVHD represents a grave outcome of allogeneic hematopoietic stem cell transplantation (HSCT), a potentially curative intervention for hematological diseases. The procedure requires radio/chemotherapy conditioning of the prospective marrow recipient, a cytotoxic process that causes vast tissue injury and, in some formats, interferes with liver production of AAT. To date, changes in the functional profile of AAT during allogeneic HSCT, and during the cytotoxic intervention that precedes HSCT, are unknown. The present study followed 53 patients scheduled for allogeneic HSCT (trial registration NCT03188601). Serum samples were tested before and after HSCT for AAT and CRP levels and for intrinsic anti-proteolytic activity. The ex vivo response to clinical-grade AAT was tested on circulating patient leukocytes and on a human epithelial cell line treated with patient sera in a gap closure assay. According to the ex vivo experiments, circulating leukocytes responded to AAT with a favorable immune-regulated profile, and epithelial gap closure was enhanced by AAT in sera from GVHD-free patients but not in sera from patients who developed GVHD. According to serum collected prior to HSCT, non-relapse mortality was reliably predicted by combining three components: AAT and CRP levels and serum anti-proteolytic activity. Taken together, HSCT outcomes are significantly affected by the anti-proteolytic function of circulating AAT, supporting early AAT augmentation therapy for allogeneic HSCT patients.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Humanos , Estudios Prospectivos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Serina Proteasas , Serina Endopeptidasas , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & controlRESUMEN
Wound healing requires a non-compromising combination of inflammatory and anti-inflammatory processes. Human α1-antitrypsin (hAAT), a circulating glycoprotein that rises during acute-phase responses and during healthy pregnancies, is tissue-protective and tolerance-inducing; although anti-inflammatory, hAAT enhances revascularization. hAAT blocks tissue-degrading enzymes, including neutrophil elastase; it is, therefore, unclear how wound healing might improve under hAAT-rich conditions. Here, wound healing was examined in the presence of recombinant hAAT (hAATWT) and protease-inhibition-lacking hAAT (hAATCP). The impact of both hAAT forms was determined by an epithelial cell gap closure assay, and by excisional skin injuries via a microemulsion optimized for open wounds. Neutrophilic infiltration was examined after 8 h. According to results, both hAAT forms accelerated epithelial gap closure and excisional wound closure, particularly at early time points. Unlike dexamethasone-treated wounds, both resulted in closed borders at the 8-h time point. In untreated and hAATCP-treated wounds, leukocytic infiltrates were widespread, in hAATWT-treated wounds compartmentalized and in dexamethasone-treated wounds, scarce. Both hAAT forms decreased interleukin-1ß and increased VEGF gene expression. In conclusion hAAT improves epithelial cell migration and outcomes of in vivo wounds irrespective of protease inhibition. While both forms of hAAT allow neutrophils to infiltrate, only native hAAT created discrete neutrophilic tissue clusters.
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Neutrófilos , Cicatrización de Heridas , Dexametasona , HumanosRESUMEN
α1-antitrypsin (AAT) is an acute-phase protein that functions as an inhibitor of serine proteases, such as neutrophil elastase. A significant body of evidence shows that AAT has a pivotal role in protecting tissues from neutrophil-induced damage, preserving endothelial function, and improving outcomes of cardiovascular and cerebrovascular diseases, though the mechanism of its activity is not fully elucidated. In terms of several significant anti-inflammatory and immunomodulatory properties, AAT's capacity to inhibit elastase has been determined to be non-essential. With the discovery of a role for diverse binding partners in AAT biology, it is intriguing to learn that AAT attaches to cholesterol, fatty acids, lipid rafts, and lipoproteins and conformational changes in its hydrophobic binding site affect its activity. The complex formation of fatty acids or lipoproteins with native or modified AAT impacts its protease inhibitory action as well as its anti-inflammatory properties. As such, AAT-enriched HDL particles exhibit improved anti-inflammatory action. These findings contribute to our understanding of AAT's mechanism of action and may provide a fresh platform for investigating the therapeutic significance of AAT therapy in an informed manner outside of genetic AAT deficiency.
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Neutrófilos , alfa 1-Antitripsina , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Ácidos Grasos , Lípidos , Pulmón/metabolismo , Neutrófilos/metabolismo , alfa 1-Antitripsina/metabolismoRESUMEN
Human α1-antitrypsin (hAAT) has two distinguishing functions: anti-protease activity and regulation of the immune system. In the present study we hypothesized that those two protein functions are mediated by different structural domains on the hAAT surface. Indeed, such biologically active immunoregulatory sites (not associated with canonical anti-protease activity) on the surface of hAAT were identified by in silico methods. Several peptides were derived from those immunoregulatory sites. Four peptides exhibited impressive biological effects in pharmacological concentration ranges. Peptidomimetic (14) was developed, based on the structure of the most druggable and active peptide. The compound exhibited a potent anti-inflammatory activity in vitro and in vivo. Such a compound could be used as a basis for developing novel anti-inflammatory drug candidates and as a research tool for better understanding hAAT functions.
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Antiinflamatorios/farmacología , Desarrollo de Medicamentos , Peptidomiméticos/farmacología , alfa 1-Antitripsina/metabolismo , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Peptidomiméticos/síntesis química , Peptidomiméticos/química , Relación Estructura-Actividad , alfa 1-Antitripsina/químicaRESUMEN
Skeletal muscle insulin resistance is a main defect in type 2 diabetes (T2D), which is associated with impaired function and content of glucose transporter type 4 (GLUT4). GLUT4 overexpression in skeletal muscle tissue can improve glucose homeostasis. Therefore, we created an engineered muscle construct (EMC) composed of GLUT4-overexpressing (OEG4) cells. The ability of the engineered implants to reduce fasting glucose levels was tested in diet-induced obesity mice. Decrease and stabilization of basal glucose levels were apparent up to 4 months after implantation. Analysis of the retrieved constructs showed elevated expression of myokines and proteins related to metabolic processes. In addition, we validated the efficiency of OEG4-EMCs in insulin-resistant mice. Following high glucose load administration, mice showed improved glucose tolerance. Our data indicate that OEG4-EMC implant is an efficient mode for restoring insulin sensitivity and improving glucose homeostasis in diabetic mice. Such procedure is a potential innovative modality for T2D therapy.
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Dendritic cells (DCs) mature upon an inflammatory trigger. However, an inflammatory trigger can lead to a semi-mature phenotype, allowing DCs to evoke tolerance and expedite the resolution of inflammation. This duality likely involves context-dependent modulation of inflammatory signaling. Human α1-antitrypsin (hAAT) promotes semimature DCs. We examined changes in a wide spectrum of signaling cascades in stimulated murine bone marrow-derived cells with hAAT. Upon stimulation by IL-1ß+IFNγ, hAAT-treated cells depicted an attenuated calcium flux. Disrupting PKA or NF-κB pathways revoked only some hAAT-mediated outcomes. hAAT-treated cells exhibited a distict pattern of kinase phosphorylation. hAAT-mediated increase in Treg cells in-vitro required intact inflammatory signaling pathways. Taken together, hAAT appears to require a stimulated microenvironment to promote inflammatory resolution, setting it aside from classical anti-inflammatory agents. Further studies are required to identify the specific molecules targeted by hAAT that mediate these and other outcomes.
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Células Dendríticas/metabolismo , Transducción de Señal/efectos de los fármacos , alfa 1-Antitripsina/farmacología , Animales , Médula Ósea/metabolismo , Células de la Médula Ósea/metabolismo , Calcio/metabolismo , Células Cultivadas , Tolerancia Inmunológica/inmunología , Inflamación/metabolismo , Interleucina-1/inmunología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Receptores CCR7/inmunología , Receptores CCR7/metabolismo , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , alfa 1-Antitripsina/metabolismoRESUMEN
Transient vestibular organ deafferentation, such that is caused by traumatic tissue injury, is presently addressed by corticosteroid therapy. However, restoration of neurophysiological properties is rarely achieved. Here, it was hypothesized that the tissue-protective attributes of α1-antityrpsin (AAT) may promote restoration of neuronal function. Inner ear injury was inflicted by unilateral labyrinthotomy in wild-type mice and in mice overexpressing human AAT. A 2-week-long assessment of vestibular signs followed. All animals responded with peak vestibular dysfunction scores within 4â¯h after local trauma. While wild-type animals displayed partial or no recovery across 7â¯days post-injury, AAT-rich group exhibited early recovery: from behavioral score 9-out-of-9 at peak to 4.8⯱â¯0.44 (mean⯱â¯SD) within 8â¯h from injury, a time when wild-type mice scored 8.6⯱â¯0.54 (pâ¯<â¯0.0001), and from vestibular score 15-out-of-15 to 7.8⯱â¯2.2 within 24â¯h, when wild-type mice scored 13.0⯱â¯2.0 (pâ¯<â¯0.01). Thus, recovery and functional normalisation of an injured vestibular compartment is achievable without corticosteroid therapy; expedited tissue repair processes appear to result from elevated circulating AAT levels. This study lays the foundation for exploring the molecular and cellular mediators of AAT within the repair processes of the delicate microscopic structures of the vestibular end organ.
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Vestíbulo del Laberinto/fisiopatología , Cicatrización de Heridas/fisiología , alfa 1-Antitripsina/metabolismo , Animales , Modelos Animales de Enfermedad , Oído Interno/lesiones , Oído Interno/fisiopatología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Vestíbulo del Laberinto/lesiones , alfa 1-Antitripsina/fisiologíaRESUMEN
RATIONALE: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. METHODS: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. RESULTS: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. CONCLUSION: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.
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Alpha1-antitrypsin (AAT) is a serum protease inhibitor that rises during inflammation and healthy pregnancies. Plasma-derived AAT, indicated for genetic AAT deficiency, is presently being explored for additional medical indications. Unlike corticosteroids, some anti-inflammatory activities of AAT involve NF-κB-dependent outcomes, e.g., induction of IL-1R antagonist. AAT activities were compared to dexamethasone (DEX), using various in-vitro cells assays, animal studies, and NF-κB-p65 localization and activity studies. Results demonstrate a cytokine shift towards resolution in AAT-treated cells, as opposed to pan-suppression in DEX-treated cells. AAT enhanced, while DEX suppressed LPS-induced IL-1Ra production and re-epithelialization. When drugs were combined, AAT allowed the immunosuppressive DEX activities, while DEX at medium to high levels antagonized beneficial AAT effects. Interestingly, lower levels of DEX maintained the immunosuppressive effect, while allowing upregulation of IL-1Ra. Therefore, AAT may represent a distinct endogenous anti-inflammatory, resolution-promoting agent that may improve tissue well-being while preventing undesired corticostroids side effects.
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Corticoesteroides/metabolismo , Inflamación/metabolismo , alfa 1-Antitripsina/metabolismo , Células A549 , Corticoesteroides/fisiología , Animales , Antiinflamatorios/farmacología , Citocinas/metabolismo , Dexametasona/metabolismo , Dexametasona/farmacología , Humanos , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Ratones , FN-kappa B/metabolismo , Células RAW 264.7 , Transducción de Señal/genética , Células THP-1 , Factor de Necrosis Tumoral alfa/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/farmacología , alfa 1-Antitripsina/fisiologíaRESUMEN
Primarily known as an elastase inhibitor, human alpha1-antitrypsin also exerts anti-inflammatory and immunomodulatory effects, both in vitro and in vivo. While the anti-protease mechanism of alpha1-antitrypsin is attributed to a particular protein domain coined the reactive center loop, anti-inflammatory and immunomodulatory loci within the molecule remain to be identified. In the present study, directed evolution and back-to-consensus algorithms were applied to human alpha1-antitrypsin. Six unique functional candidate sites were identified on the surface of the molecule; in manipulating these sites by point mutations, a recombinant mutant form of alpha1-antitrypsin was produced, depicting a requirement for sites outside the reactive center loop as essential for protease inhibition, and displaying enhanced anti-inflammatory activities. Taken together, outcomes of the present study establish a potential use for directed evolution in advancing our understanding of site-specific protein functions, offering a platform for development of context- and disease-specific alpha1-antitrypsin-based therapeutics.
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alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Algoritmos , Animales , Antiinflamatorios , Evolución Molecular Dirigida/métodos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Péptido Hidrolasas/metabolismo , Proteolisis , alfa 1-Antitripsina/ultraestructuraRESUMEN
OBJECTIVES: To determine the levels of endogenous α1-antitrypsin in the perilymph of patients undergoing cochlear implant (CI), and its reverse association with the severity of hearing loss. STUDY DESIGN: Retrospective study. SETTING: Tertiary care university hospital. PARTICIPANTS: The study includes 38 patients undergoing CI surgery, 11 patients diagnosed with congenital deafness and 27 non-congenital deafness, eight patients diagnosed with moderate hearing loss (N = 8; PTA = 70 dB), severe hearing loss (N = 11; PTA 70-90 dB) and profound hearing loss (N = 19; PTA > 90 dB). MAIN OUTCOME AND MEASURE: 1 to 12 µL perilymphatic fluids were collected by micropipette. α1-antitrypsin levels were determined, and current and historic audiological parameters were obtained. RESULTS: The congenital and non-congenital group exhibited AAT concentrations of 2.5 ± 1.9 × 106 LFQ and 3.2 ± 1.2 × 106 LFQ, respectively (mean ± SD; P = .38). Mean levels of α1-antitrypsin in the perilymph fluid within the moderate group was 3.64 × 106 ± 2.1 × 106 LFQ vs 3.5 × 106 ± 1.2 × 106 in severe hearing loss (P = .81) and 2.4 × 106 ± 1.1 × 106 LFQ in the profound hearings loss group (P = .06). The difference in levels of AAT in samples from the severe hearings loss group vs the profound hearings loss group reached statistical significance (P = .04). CONCLUSION: Insufficiency in α1-antitrypsin levels in the perilymph fluid of the inner ear appears to display a relationship with the severity of hearing loss. The prospect of introducing clinical-grade plasma-purified α1-antitrypsin directly onto the site of cochlear injury deserves thorough investigation.
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Pérdida Auditiva/cirugía , Fragmentos de Péptidos/metabolismo , Perilinfa/química , alfa 1-Antitripsina/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Pérdida Auditiva/congénito , Humanos , Lactante , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Índice de Severidad de la EnfermedadRESUMEN
Mitochondrial turnover is required for proper cellular function. Both mitochondrial biogenesis and mitophagy are impaired in several degenerative and age-related diseases. The search for mitophagy activators recently emerged as a new therapeutical approach; however, there is a lack in suitable tools to follow mitochondrial turnover in a high-throughput manner. We demonstrate that the fluorescent protein, MitoTimer, is a reliable and robust probe to follow mitochondrial turnover. The screening of 15 000 small molecules led us to two chemically-related benzothiophenes that stimulate basal mitophagy in the beta-cell line, INS1. Enhancing basal mitophagy was associated with improved mitochondrial function, higher Complex I activity and Complex II and III expressions in INS1 cells, as well as better insulin secretion performance in mouse islets. The possibility of further enhancing mitophagy in the absence of mitochondrial stressors points to the existence of a 'basal mitophagy spare capacity'. To this end, we found two small molecules that can be used as models to better understand the physiological regulation of mitophagy.
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Envejecimiento/genética , Secreción de Insulina/genética , Mitocondrias/genética , Mitofagia/genética , Envejecimiento/patología , Animales , Autofagia/genética , Línea Celular , Citometría de Flujo , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Recambio Mitocondrial , Mitofagia/efectos de los fármacos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Tiofenos/química , Tiofenos/farmacologíaRESUMEN
Immunosuppressive drugs are an inherent component of hematopoietic stem cell transplantation (HSCT) for the prevention of acute graft-versus-host disease (GVHD). Circulating α1-antitrypsin (AAT), a serine-protease inhibitor produced predominantly by hepatocytes that rises during acute phase responses, is lost in patient's stool due to gastrointestinal GVHD, and its augmentation has been found to attenuate GVHD. Here we explored the effect of immunosuppressive drugs on hepatocyte production of AAT and intestinal epithelial gap repair. The effect of commonly used immunosuppressants on AAT production was examined in vitro using HepG2 cells and primary mouse hepatocytes, and their impact on human intestinal epithelial cell line gap repair was evaluated. Sera from 12 allogeneic HSCT recipients, obtained at 14 days post-transplantation, predating the diagnosis of GVHD (n = 6), were examined for reepithelialization, with added clinical-grade AAT. Rapamycin compromised AAT production under inflammatory conditions. Mycophenolate mofetil and cyclosporine A (CSA) inhibited reepithelialization; AAT minimized the effect of CSA. Patient sera displayed superior gap repair with exogenous AAT. Functional insufficiency in circulating AAT may be the result of drug toxicities leading to ineffective gut reepithelization and compromised gut lining. Taken together, our data strengthen the rationale for incorporating AAT augmentation therapy into immunosuppressive treatment protocols.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Preparaciones Farmacéuticas , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/prevención & control , Hepatocitos , Humanos , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Ácido MicofenólicoRESUMEN
Genetic aberrations in the toll-like receptor (TLR)3 pathway are associated with increased susceptibility to herpes simplex virus (HSV) infections. Leucine-rich repeat and PYD-containing protein (NLRP)12 is a component of the inflammasome apparatus, which is critical to an immediate innate inflammatory response. Aberrations in NLRP12 have been shown to mediate auto-inflammation. In this study, we present a 44-year old patient with severe HSV esophagitis and Crohn's disease. An immune and genetic investigation confirmed two coinciding genetic mutations in TLR3 and NLRP12. Our findings support conducting laboratory workup that targets TLR3 pathway in the immunocompetent host developing recurrent HSV infections.
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Enfermedad de Crohn/genética , Esofagitis/genética , Herpes Simple/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Receptor Toll-Like 3/genética , Aciclovir/uso terapéutico , Adulto , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antivirales/uso terapéutico , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/inmunología , Esofagitis/inmunología , Esofagitis/virología , Femenino , Fármacos Gastrointestinales/uso terapéutico , Herpes Simple/tratamiento farmacológico , Herpes Simple/inmunología , Herpes Simple/prevención & control , Humanos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Mutación , Transducción de Señal , Receptor Toll-Like 3/inmunología , Valganciclovir/uso terapéutico , Secuenciación Completa del GenomaRESUMEN
Our aim was to assess the efficacy, safety, and tolerability of alpha-1 antitrypsin (AAT) as a therapeutic modality for ß-cell preservation in patients with recent-onset type 1 diabetes. Seventy type 1 diabetes patients (37 males; mean age 13.1 ± 4.1years) were randomized to treatment with 22 infusions of AAT (Glassia®) (60 or 120 mg/kg) or placebo. The primary outcome was the area under the curve (AUC) of C-peptide from a 2-h mixed-meal tolerance test after 52 weeks. At week 52, C-peptide was 0.9, 0.45, and 0.48 pmol/mL in the AAT-120, AAT-60, and placebo groups (p = 0.170 and p = 0.866 vs. placebo, respectively). The declines in C-peptide glycated hemoglobin (HbA1c) and the total insulin dose (U/kg) were similar across groups. Within the predefined 12-18-years subgroup, the C-peptide AUC decreased significantly in the placebo and AAT-60 groups (-0.34 and -0.54 pmol/mL, respectively, p < 0.01), with a borderline decrease in the AAT-120 group (-0.29 pmol/mL, p = 0.047). The mean HbA1c level was significantly lower in the AAT-120 group compared to the placebo (6.7% ± 0.9% vs. 8.2 ± 1.4%, p = 0.05), and a higher percentage of patients attained HbA1c ≤ 7% (75% vs. 25%, p = 0.05). AAT was tolerated well, with a similar safety profile between groups. The AAT intervention showed promise in the subgroup of adolescents with recent-onset type 1 diabetes. Further studies are warranted to determine the impact and proposed mechanism of action of AAT in ß-cell preservation.
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Diabetes Mellitus Tipo 1/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , alfa 1-Antitripsina/uso terapéutico , Adolescente , Adulto , Niño , Diabetes Mellitus Tipo 1/patología , Método Doble Ciego , Femenino , Humanos , Factores Inmunológicos/efectos adversos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Masculino , Efecto Placebo , Resultado del Tratamiento , Adulto Joven , alfa 1-Antitripsina/efectos adversosRESUMEN
Objective: Skin flaps are routinely used in reconstructive surgery yet remain susceptible to ischemia and necrosis. Distant flaps require lengthy time to detach causing patient discomfort. Human α1-antitrypsin (hAAT) is a clinically available serum glycoprotein. hAAT was shown to support mature vessel formation and enhance tissue survival following ischemia-reperfusion injuries. The purpose of the presented study was to examine the effect of hAAT on skin flap survival and distant "tube" flap perfusion through its recipient site. Approach: Random-pattern skin flaps were performed on mice treated with clinical-grade hAAT using three unique routes of administration (transgenic, i.p. and s.c. infiltration); necrotic area and tissue perfusion were assessed. Blockade of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) were used to explore aspects of mechanism of action. A distant tube flap model was performed to examine time to perfusion. Results: hAAT-treated mice displayed approximately two-fold smaller necrotic flap areas versus controls across all hAAT administration routes. Flaps displayed greater perfusion as early as 3 days postsurgery (64.6% ± 4.0% vs. 43.7% ± 1.7% in controls; p = 0.007). hAAT-mediated flap survival was prominently NOS dependent, but only partially VEGF dependent. Finally, distant flaps treated with hAAT displayed significantly earlier perfusion versus controls (mean 9.6 ± 1.6 vs. 13.1 ± 1.0 days; p = 0.0005). Innovation: The established safety record of hAAT renders it an attractive candidate toward improving skin flap surgery outcomes, particularly during VEGF blockade. Conclusions: hAAT treatment enhances survival and accelerates perfusion of skin flaps in animal models in a NOS-dependent manner, partially circumventing VEGF blockade. Further mechanistic studies are required.
RESUMEN
Background: Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. in vivo, hAAT reduces bacterial load, without directly inhibiting bacterial growth. In conditions of excess nitric-oxide (NO), hAAT undergoes S-nitrosylation (S-NO-hAAT) and gains antibacterial capacity. The impact of S-NO-hAAT on immune cells has yet to be explored. Aim: Study the effects of S-NO-hAAT on immune cells during bacterial infection. Methods: Clinical-grade hAAT was S-nitrosylated and then compared to unmodified hAAT, functionally, and structurally. Intracellular bacterial clearance by THP-1 macrophages was assessed using live Salmonella typhi. Murine peritoneal macrophages were examined, and signaling pathways were evaluated. S-NO-hAAT was also investigated after blocking free mambranal cysteine residues on cells. Results: S-NO-hAAT (27.5 uM) enhances intracellular bacteria elimination by immunocytes (up to 1-log reduction). S-NO-hAAT causes resting macrophages to exhibit a pro-inflammatory and antibacterial phenotype, including release of inflammatory cytokines and induction of inducible nitric oxide synthase (iNOS) and TLR2. These pro-inflammatory effects are dependent upon cell surface thiols and activation of MAPK pathways. Conclusions: hAAT duality appears to be context-specific, involving S-nitrosylation in a nitric oxide rich environment. Our results suggest that S-nitrosylation facilitates the antibacterial activity of hAAT by promoting its ability to activate innate immune cells. This pro-inflammatory effect may involve transferring of nitric oxide from S-NO-hAAT to a free cysteine residue on cellular targets.