RESUMO
Bacillus anthracis and Yersinia pestis, causative pathogens for anthrax and plague, respectively, along with Burkholderia mallei and B. pseudomallei are potential bioterrorism threats. Tebipenem pivoxil hydrobromide (TBP HBr, formerly SPR994), is an orally available prodrug of tebipenem, a carbapenem with activity versus multidrug-resistant (MDR) gram-negative pathogens, including quinolone-resistant and extended-spectrum-ß-lactamase-producing Enterobacterales. We evaluated the in vitro activity and in vivo efficacy of tebipenem against biothreat pathogens. Tebipenem was active in vitro against 30-strain diversity sets of B. anthracis, Y. pestis, B. mallei, and B. pseudomallei with minimum inhibitory concentration (MIC) values of 0.001 - 0.008 µg/ml for B. anthracis, ≤0.0005 - 0.03 µg/ml for Y. pestis, 0.25 - 1 µg/ml for B. mallei, and 1 - 4 µg/ml for B. pseudomallei In a B. anthracis murine model, all control animals died within 52 h post challenge. The survival rates in the groups treated with tebipenem were 75% and 73% when dosed at 12 h and 24 h post challenge, respectively. The survival rates in the positive control groups treated with ciprofloxacin were 75% and when dosed 12 h and 25% when dosed 24 h post challenge, respectively. Survival rates were significantly (p=0.0009) greater in tebipenem groups treated at 12 h and 24 h post challenge and in the ciprofloxacin group 12 h post-challenge vs. the vehicle-control group. For Y. pestis, survival rates for all animals in the tebipenem and ciprofloxacin groups were significantly (p<0.0001) greater than the vehicle-control group. These results support further development of tebipenem for treating biothreat pathogens.
RESUMO
Marfan syndrome (MFS) is an autosomal dominant genetic disorder caused by mutations in the FBN1 gene. Although many peripheral tissues are affected, aortic complications, such as dilation, dissection and rupture, are the leading causes of MFS-related mortality. Aberrant TGF-beta signalling plays a major role in the pathophysiology of MFS. However, the contributing mechanisms are still poorly understood. Here, we aimed at identifying novel aorta-specific pathways involved in the pathophysiology of MFS. For this purpose, we employed the Fbn1 under-expressing mgR/mgR mouse model of MFS. We performed RNA-sequencing of aortic tissues of 9-week-old mgR/mgR mice compared with wild-type (WT) mice. With a false discovery rate <5%, our analysis revealed 248 genes to be differentially regulated including 20 genes previously unrelated with MFS-related pathology. Among these, we identified Igfbp2, Ccl8, Spp1, Mylk2, Mfap4, Dsp and H19. We confirmed the expression of regulated genes by quantitative real-time PCR. Pathway classification revealed transcript signatures involved in chemokine signalling, cardiac muscle contraction, dilated and hypertrophic cardiomyopathy. Furthermore, our immunoblot analysis of aortic tissues revealed altered regulation of pSmad2 signalling, Perk1/2, Igfbp2, Mfap4, Ccl8 and Mylk2 protein levels in mgR/mgR vs WT mice. Together, our integrative systems approach identified several novel factors associated with MFS-aortic-specific pathophysiology that might offer potential novel therapeutic targets for MFS.
Assuntos
Aorta Torácica/metabolismo , Proteínas de Transporte/genética , Proteínas da Matriz Extracelular/genética , Fibrilina-1/genética , Glicoproteínas/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Síndrome de Marfan/genética , Osteopontina/genética , Animais , Aorta Torácica/fisiopatologia , Proteínas de Transporte/metabolismo , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/metabolismo , Fibrilina-1/deficiência , Regulação da Expressão Gênica , Ontologia Genética , Glicoproteínas/metabolismo , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Síndrome de Marfan/metabolismo , Síndrome de Marfan/fisiopatologia , Camundongos , Camundongos Transgênicos , Anotação de Sequência Molecular , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Osteopontina/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Biologia de Sistemas/métodos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.
Assuntos
Inativação Gênica , Terapia Genética , Proteínas de Homeodomínio/genética , Morfolinos/genética , Distrofia Muscular Facioescapuloumeral/genética , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Marcação de Genes , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Morfolinos/administração & dosagem , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Distrofia Muscular Facioescapuloumeral/terapia , TranscriptomaRESUMO
OBJECTIVE: Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of noncanonical (Erk-mediated) TGFß signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFß neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFß neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1(mgR/mgR) mice). APPROACH AND RESULTS: Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1(mgR/mgR) mice were compared after a ≈3-month-long treatment with placebo and either the AT1r antagonist losartan or the TGFß-neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFß neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (postnatal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence-related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFß hyperactivity that was in part AT1r dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1(mgR/mgR) mice from ruptured TAA. CONCLUSIONS: By demonstrating that promiscuous AT1r and TGFß drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways although sparing the early protective role of TGFß.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Anticorpos Neutralizantes/farmacologia , Aorta Torácica/efeitos dos fármacos , Aneurisma da Aorta Torácica/prevenção & controle , Losartan/farmacologia , Síndrome de Marfan/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Ruptura Aórtica/genética , Ruptura Aórtica/metabolismo , Ruptura Aórtica/patologia , Ruptura Aórtica/prevenção & controle , Modelos Animais de Doenças , Progressão da Doença , Fibrilina-1 , Fibrilinas , Humanos , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas dos Microfilamentos/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mutação , Fosforilação , Receptor Tipo 1 de Angiotensina/metabolismo , Proteína Smad2/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Respiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-ß play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-ß signaling improves respiratory function, mdx mice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-ß), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdx mice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-ß antagonism may be a useful therapeutic approach for treating DMD patients.
Assuntos
Respiração , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Creatina Quinase/sangue , Diafragma/efeitos dos fármacos , Diafragma/metabolismo , Diafragma/patologia , Diafragma/fisiopatologia , Relação Dose-Resposta a Droga , Enalapril/administração & dosagem , Enalapril/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Força da Mão/fisiologia , Hidroxiprolina/metabolismo , Inflamação/sangue , Inflamação/metabolismo , Inflamação/patologia , Losartan/administração & dosagem , Losartan/farmacologia , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Miogenina/metabolismo , Tamanho do Órgão/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Respiração/efeitos dos fármacos , Testes de Função Respiratória , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Exposure to acute, high-dose, whole-body ionizing radiation results in bone marrow failure (hematopoietic acute radiation syndrome with resultant infection, bleeding, anemia, and increased risk of death). Sargramostim (yeast-derived rhu GM-CSF), a yeast-derived, molecularly cloned, hematopoietic growth factor and pleiotropic cytokine supports proliferation, differentiation, maturation and survival of cells of several myeloid lineages. We evaluated the efficacy of sargramostim in non-human primates (rhesus macaques) exposed to whole-body ionizing radiation at a 50-60% lethal dose. The primary end point was day 60 survival. Non-human primates received daily subcutaneous sargramostim (7 mcg/kg/day) or control. To reflect the anticipated setting of a nuclear or radiologic event, treatment began 48 h postirradiation, and non-human primates received only moderate supportive care (no whole blood transfusions or individualized antibiotics). Sargramostim significantly increased day 60 survival to 78% (95% confidence interval, 61-90%) vs. 42% (26-59%; P = 0.0018) in controls. Neutrophil, platelet and lymphocyte recovery rates were accelerated and infection rates decreased. Improved survival when sargramostim was started 48 h postirradiation, without use of intensive supportive care, suggests sargramostim may be effective in treating humans exposed to acute, high-dose whole-body, ionizing radiation in a scenario such as a mass casualty event.
Assuntos
Síndrome Aguda da Radiação/tratamento farmacológico , Células da Medula Óssea/efeitos dos fármacos , Transtornos da Insuficiência da Medula Óssea/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Síndrome Aguda da Radiação/genética , Síndrome Aguda da Radiação/patologia , Animais , Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/efeitos da radiação , Transtornos da Insuficiência da Medula Óssea/genética , Transtornos da Insuficiência da Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Macaca mulatta/genética , Masculino , Proteínas Recombinantes/farmacologia , Irradiação Corporal Total/efeitos adversosRESUMO
A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFß modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1(Prx1-/-) mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1(Prx1-/-) mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1(Prx1-/-) mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin-1 modulation of TGFß bioavailability, cultures of marrow stromal cells from Fbn1(Prx1-/-) limb bones showed improper overactivation of latent TGFß. In line with this finding, systemic TGFß neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFß bioavailability within the microenvironment of marrow niches.
Assuntos
Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Nicho de Células-Tronco/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Fibrilina-1 , Fibrilinas , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.
RESUMO
Expansions of CUG trinucleotide sequences in RNA transcripts provide the basis for toxic RNA gain-of-function that leads to detrimental changes in RNA metabolism. A CTG repeat element normally resides in the 3' untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene, but when expanded it is the genetic lesion of myotonic dystrophy type 1 (DM1), a hereditary neuromuscular disease. The pathogenic DMPK transcript containing the CUG expansion is retained in ribonuclear foci as part of a complex with RNA-binding proteins such as muscleblind-like 1 (MBNL1), resulting in aberrant splicing of numerous RNA transcripts and consequent physiological abnormalities including myotonia. Herein, we demonstrate molecular and physiological amelioration of the toxic effects of mutant RNA in the HSA(LR) mouse model of DM1 by systemic administration of peptide-linked morpholino (PPMO) antisense oligonucleotides bearing a CAG repeat sequence. Intravenous administration of PPMO conjugates to HSA(LR) mice led to redistribution of Mbnl1 protein in myonuclei and corrections in abnormal RNA splicing. Additionally, myotonia was completely eliminated in PPMO-treated HSA(LR) mice. These studies provide proof of concept that neutralization of RNA toxicity by systemic delivery of antisense oligonucleotides that target the CUG repeat is an effective therapeutic approach for treating the skeletal muscle aspects of DM1 pathology.
Assuntos
Morfolinos/administração & dosagem , Distrofia Miotônica/genética , Peptídeos/administração & dosagem , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas/genética , Animais , Humanos , Camundongos , Morfolinos/química , Mutação , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Miotonina Proteína Quinase , Oligonucleotídeos Antissenso/administração & dosagem , Peptídeos/química , Proteínas Serina-Treonina Quinases/genética , RNA/genética , RNA/toxicidade , Splicing de RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genéticaRESUMO
The purpose of this study was to determine whether inhibition of potassium channels or cytochrome P450 attenuates the transient phase of hypotension during endotoxic shock in vivo, and to determine whether these interventions improve the rate of survival. Male Sprague-Dawley rats were pretreated with saline (0.2 ml, i.v.), tetraethylammonium chloride (TEA 30 mg/kg; 0.2 ml, i.v.), proadifen (SKF-525 A; 50 mg/kg, i.p.) or ketoconazole (50 mg/kg, i.p.) and challenged with lipopolysaccharide (LPS; 20 mg/kg, i.p.). Changes in heart rate, mean (MAP), systolic (SP) and diastolic (DP) arterial pressures as well as survival rate were then monitored for 45 min. Potassium channel inhibition with TEA had no effect on LPS-induced hypotension at any time point compared with saline (maximal fall in MAP of 79 +/- 18 and 80 +/- 13 mm Hg, respectively). Pretreatment with proadifen or ketoconazole, inhibitors of cytochrome P450, significantly attenuated LPS-induced hypotension compared with saline (maximal fall in MAP of 34, 26 and 63% below baseline, respectively). This effect was evident in all arterial pressures measured, MAP, SP and DP. At 45 min, the survival rate in the saline group was 66%. Pretreatment with TEA significantly reduced survival rate to 50% and pretreatment with proadifen or ketoconazole improved survival to 100% (p < 0.05). These results suggest that an arachidonic acid metabolite produced by a cytochrome P450-catalyzed reaction may contribute to the transient phase of LPS-induced hypotension. However, these effects do not appear to be mediated through potassium channel activation.