RESUMO
Pulmonary ischemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centered NK cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. We hypothesized that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.
RESUMO
Chemotherapy of multi-drug-resistant tuberculosis (TB) requires prolonged administration of multiple drugs. We investigated whether pulmonary delivery of minute doses of drugs, along with reduced oral doses of the same agents, would affect preclinical efficacy. We prepared dry powder inhalation (DPI) formulations comprising sutezolid (SUT), the second-generation pretomanid analog TBA-354 (TBA), or a fluorinated derivative of TBA-354 (32,625) in a matrix of the biodegradable polymer poly(L-lactide). We established formulation characteristics, doses inhaled by healthy mice, and preclinical efficacy in a mouse model of TB. Oral doses of 100 mg/kg/day or DPI doses of 0.25-0.5 mg/kg/day of drugs SUT, TBA-354, or 32,625 administered over 28 days were sub-optimally effective in reducing lung and spleen burden of Mycobacterium tuberculosis (Mtb) in infected mice. The addition of 0.25-0.5 mg/kg/day of SUT, TBA-354, or 32,625 as DPI to oral doses of 50 mg/kg/day was non-inferior in clearing Mtb from the lungs of infected mice. We concluded that adjunct therapy with inhaled second-line agents has the potential to reduce the efficacious oral dose.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Animais , Camundongos , Antituberculosos , Preparações Farmacêuticas , Redução da Medicação , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Administração por Inalação , PósRESUMO
BACKGROUND: It is unclear whether Vitamin D is efficacious as a host-directed therapy (HDT) for patients of tuberculosis (TB). We investigated pulmonary delivery of the active metabolite of Vitamin D3, i.e., 1, 25-dihydroxy vitamin D3 (calcitriol) in a mouse model of infection with Mycobacterium tuberculosis (Mtb). METHODS: We optimized a spray drying process to prepare a dry powder inhalation (DPI) of calcitriol using a Quality by Design (QbD) approach. We then compared outcomes when Mtb-infected mice were treated with inhaled calcitriol at 5 ng/kg as a stand-alone intervention versus DPI as adjunct to standard oral anti-tuberculosis therapy (ATT). RESULTS: The DPI with or without concomitant ATT markedly improved the morphology of the lungs and mitigated histopathology in both the lungs and the spleens. The number of nodular lesions on the lung surface decreased from 43.7 ± 3.1 to 22.5 ± 3.9 with the DPI alone and to 9.8 ± 2.5 with DPI + ATT. However, no statistically significant induction of host antimicrobial peptide cathelicidin or reduction in bacterial burden was seen with the DPI alone. DPI + ATT did not significantly reduce the bacterial burden in the lungs compared to ATT alone. CONCLUSIONS: We concluded that HDT using the low dose calcitriol DPI contributed markedly to mitigation of pathology, but higher dose may be required to evoke significant induction of bactericidal host response and bactericidal activity in the lung.
Assuntos
Calcitriol , Tuberculose , Administração por Inalação , Animais , Antituberculosos/farmacologia , Calcitriol/farmacologia , Inaladores de Pó Seco , Camundongos , Pós , Tuberculose/tratamento farmacológicoRESUMO
We compared the pharmacokinetics and efficacy of a combination of d-cycloserine (DCS) and ethionamide (ETO) via oral and inhalation routes in mice. The plasma half-life (t1/2) of oral ETO at a human-equivalent dose decreased from 4.63 ± 0.61 h to 1.64 ± 0.40 h when DCS was coadministered. The area under the concentration-time curve from 0 h to time t (AUC0-t ) was reduced to one-third. Inhalation overcame the interaction. Inhalation, but not oral doses, reduced the lung CFU/g of Mycobacterium tuberculosis H37Rv from 6 to 3 log10 in 4 weeks, indicating bactericidal activity.
Assuntos
Antituberculosos/farmacocinética , Ciclosserina/farmacocinética , Etionamida/farmacocinética , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Pulmonar/tratamento farmacológico , Administração por Inalação , Administração Oral , Animais , Antituberculosos/administração & dosagem , Ciclosserina/administração & dosagem , Farmacorresistência Bacteriana , Etionamida/administração & dosagem , Pulmão/microbiologia , Camundongos , Tuberculose Pulmonar/microbiologiaRESUMO
Up to 90% of the global population has been infected with cytomegalovirus (CMV), a herpesvirus that remains latent for the lifetime of the host and drives immune dysregulation. CMV is a critical risk factor for poor outcomes after solid organ transplant, though lung transplant recipients (LTR) carry the highest risk of CMV infection, and CMV-associated comorbidities compared to recipients of other solid organ transplants. Despite potent antivirals, CMV remains a significant driver of chronic lung allograft dysfunction (CLAD), re-transplantation, and death. Moreover, the extended utilization of CMV antiviral prophylaxis is not without adverse effects, often necessitating treatment discontinuation. Thus, there is a critical need to understand the immune response to CMV after lung transplantation. This review identifies key elements of each arm of the CMV immune response and highlights implications for lung allograft tolerance and injury. Specific attention is paid to cellular subsets of adaptive and innate immune cells that are important in the lung during CMV infection and reactivation. The concept of heterologous immune responses is reviewed in depth, including how they form and how they may drive tissue- and allograft-specific immunity. Other important objectives of this review are to detail the emerging role of NK cells in CMV-related outcomes, in addition to discussing perturbations in CMV immune function stemming from pre-existing lung disease. Finally, this review identifies potential mechanisms whereby CMV-directed treatments may alter the cellular immune response within the allograft.
RESUMO
Mycobacterium tuberculosis (Mtb) infection leads to upregulation of Suppressors of Cytokine signaling (SOCS) expression in host macrophages (MÏ). SOCS proteins inhibit cytokine signaling by negatively regulating JAK/STAT. We investigated this host-pathogen dialectic at the level of transcription. We used phorbol-differentiated THP-1 MÏ infected with Mtb to investigate preferential upregulation of some SOCS isoforms that are known to inhibit signaling by IFN-γ, IL-12, and IL-6. We examined time kinetics of likely transcription factors and signaling molecules upstream of SOCS transcription, and survival of intracellular Mtb following SOCS upregulation. Our results suggest a plausible mechanism that involves PGE2 secretion during infection to induce the PKA/CREB axis, culminating in nuclear translocation of C/EBPß to induce expression of SOCS1. Mtb-infected MÏ secreted IL-10, suggesting a mechanism of induction of STAT3, which may subsequently induce SOCS3. We provide evidence of temporal variation in SOCS isoform exspression and decay. Small-interfering RNA-mediated knockdown of SOCS1 and SOCS3 restored the pro-inflammatory milieu and reduced Mtb viability. In mice infected with Mtb, SOCS isoforms persisted across Days 28-85 post infection. Our results suggest that differential temporal regulation of SOCS isoforms by Mtb drives the host immune response towards a phenotype that facilitates the pathogen's survival.
Assuntos
Mycobacterium tuberculosis , Humanos , Animais , Camundongos , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Macrófagos/microbiologia , Interleucina-12 , Isoformas de Proteínas/metabolismoRESUMO
Transient transfection of the respiratory mucosa of mice infected with Mycobacterium tuberculosis (Mtb) with gamma interferon (IFN-γ) promises benefits in disease therapy. We investigated preclinical efficacy of a dry powder inhalation (DPI) as a stand-alone versus adjunct to oral anti-tuberculosis (TB) chemotherapy in mice. We observed that this host-directed therapy mitigates the gross organ pathology and histopathology of lung and spleen tissue of infected mice receiving the DPI, either alone or as adjunct therapy. However, no statistically significant reduction in Mtb colony forming units (CFU) occurred if mice were given only DPI; but not drugs. We compared one and three doses a week of the DPI over four weeks; with or without concomitant oral drugs. There was no significant difference in lung CFU after four or 12 doses of the DPI alone, but, surprisingly, four doses were qualitatively better than 12 doses in mitigating lung pathology. Nodular lesions on the lung surface and the area occupied by these was significantly reduced after four doses of the DPI, even without oral drugs. Transient transfection with IFN-γ did not induce pathological inflammation of the lungs and airways. We conclude that IFN-γ, as expected of host-directed therapy, 'heals the host; ' but does not 'kill the bug.'
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Antituberculosos/uso terapêutico , Modelos Animais de Doenças , Terapia Genética , Interferon gama/genética , Pulmão/microbiologia , Camundongos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
Nebulized gamma interferon (IFN-γ) protein has been studied for clinical safety and efficacy against pulmonary tuberculosis (TB). The protein is expensive, requires a cold chain, and is difficult to deploy in limited-resource, high-incidence settings. We generated a preclinical proof of concept (PoC) for a dry powder inhalation (DPI) containing DNA constructs to transiently transfect the lung and airway epithelium of mice with murine IFN-γ. Bacterial colony-forming units (CFU) in the lungs of mice infected with Mycobacterium tuberculosis (Mtb) reduced from about 106/g of tissue to ~104 after four doses given once a week. Nodular inflammatory lesions in the lungs reduced significantly in number. Immunohistochemistry of infected lung sections for LC3-1 and LAMP-1 indicated autophagy induction between 18 and 48 h after inhalation. ELISA on bronchoalveolar lavage (BAL) fluid showed differences in kinetics of IFN-γ concentrations in the epithelial lining fluid of healthy versus infected mice. Uninfected mice receiving DNA constructs expressing a fluorescent protein were live-imaged. The fluorescence signals from the intracellular protein peaked at about 36 h after inhalation and declined by 48 h. These results establish preclinical PoC of the efficacy of a DPI and dosing regimen as a host-directed and transient gene therapy of experimental pulmonary TB in mice, justifying preclinical development.
RESUMO
A spray drying process was standardized to prepare an inhalable powder comprising d-cycloserine and ethionamide, two "second line" drugs employed for treating multi-drug resistant (MDR) tuberculosis (TB). The aim of the process development effort was to maximize product yield. Contour plots were generated using a small central composite design (CCD) with face centered (αâ¯=â¯1) to maximize the process yield as the response criterion. The design space was experimentally validated. Powder was prepared and characterized for drug content (HPLC), geometric size (laser scattering), surface morphology (scanning electron microscopy) aerosol behaviour (cascade impaction) and powder flow properties. The optimized process yielded a powder with a median mass aerodynamic diameter (MMAD) of 1.76 µâ¯±â¯3.1 geometric standard deviation (GSD). Mass balance indicated that the major proportion of the particles produced by spray drying are lost to the outlet filter. The process represents a best-case compromise of spray-drying conditions to minimize loss during droplet drying, collection and process air discharge.