Mycobacteriophages: therapeutic approach for mycobacterial infections.

Tuberculosis (TB) is a significant global health threat, and cases of infection with non-tuberculous mycobacteria (NTM) causing lung disease (NTM-LD) are rising. Bacteriophages and their gene products have garnered interest as potential therapeutic options for bacterial infections. Here, we have compiled information on bacteriophages and their products that can kill Mycobacterium tuberculosis or NTM. We summarize the mechanisms whereby viable phages can access macrophage-resident bacteria and not elicit immune responses, review methodologies of pharmaceutical product development containing mycobacteriophages and their gene products, mainly lysins, in the context of drug regulatory requirements and we discuss industrially relevant methods for producing pharmaceutical products comprising mycobacteriophages, emphasizing delivery of mycobacteriophages to the lungs. We conclude with an outline of some recent case studies on mycobacteriophage therapy.

Insights into the genomic features and lifestyle of B1 subcluster mycobacteriophages.

Bacteriophages infecting Mycobacterium smegmatis mc2155 are numerous and, hence, are classified into clusters based on nucleotide sequence similarity. Analyzing phages belonging to clusters/subclusters can help gain deeper insights into their biological features and potential therapeutic applications. In this study, for genomic characterization of B1 subcluster mycobacteriophages, a framework of online tools was developed, which enabled functional annotation of about 55% of the previously deemed hypothetical proteins in B1 phages. We also studied the phenotype, lysogeny status, and antimycobacterial activity of 10 B1 phages against biofilm and an antibiotic-resistant M. smegmatis strain (4XR1). All 10 phages belonged to the Siphoviridae family, appeared temperate based on their spontaneous release from the putative lysogens and showed antibiofilm activity. The highest inhibitory and disruptive effects on biofilm were 64% and 46%, respectively. This systematic characterization using a combination of genomic and experimental tools is a promising approach to furthering our understanding of viral dark matter.

Transcriptional regulation of suppressors of cytokine signaling during infection with Mycobacterium tuberculosis in human THP-1-derived macrophages and in mice.

Mycobacterium tuberculosis (Mtb) infection leads to upregulation of Suppressors of Cytokine signaling (SOCS) expression in host macrophages (Mϕ). 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 Mϕ 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 Mϕ 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.

Inhaled Adjunct Therapy with Second-Line Drug Candidates for Dose Reduction in Chemotherapeutic Regimens for Multi-drug-Resistant Tuberculosis.

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.

Preparation and Evaluation of Low-Dose Calcitriol Dry Powder Inhalation as Host-Directed Adjunct Therapy for Tuberculosis.

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.

Phase Transitions of Cu and Fe at Multiscales in an Additively Manufactured Cu-Fe Alloy under High-Pressure.

A state of the art, custom-built direct-metal deposition (DMD)-based additive manufacturing (AM) system at the University of Michigan was used to manufacture 50Cu-50Fe alloy with tailored properties for use in high strain/deformation environments. Subsequently, we performed preliminary high-pressure compression experiments to investigate the structural stability and deformation of this material. Our work shows that the alpha (BCC) phase of Fe is stable up to ~16 GPa before reversibly transforming to HCP, which is at least a few GPa higher than pure bulk Fe material. Furthermore, we observed evidence of a transition of Cu nano-precipitates in Fe from the well-known FCC structure to a metastable BCC phase, which has only been predicted via density functional calculations. Finally, the metastable FCC Fe nano-precipitates within the Cu grains show a modulated nano-twinned structure induced by high-pressure deformation. The results from this work demonstrate the opportunity in AM application for tailored functional materials and extreme stress/deformation applications.

Transient, inhaled gene therapy with gamma interferon mitigates pathology induced by host response in a mouse model of tuberculosis.

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.'

Compositionally-Driven Formation Mechanism of Hierarchical Morphologies in Co-Deposited Immiscible Alloy Thin Films.

Co-deposited, immiscible alloy systems form hierarchical microstructures under specific deposition conditions that accentuate the difference in constituent element mobility. The mechanism leading to the formation of these unique hierarchical morphologies during the deposition process is difficult to identify, since the characterization of these microstructures is typically carried out post-deposition. We employ phase-field modeling to study the evolution of microstructures during deposition combined with microscopy characterization of experimentally deposited thin films to reveal the origin of the formation mechanism of hierarchical morphologies in co-deposited, immiscible alloy thin films. Our results trace this back to the significant influence of a local compositional driving force that occurs near the surface of the growing thin film. We show that local variations in the concentration of the vapor phase near the surface, resulting in nuclei (i.e., a cluster of atoms) on the film's surface with an inhomogeneous composition, can trigger the simultaneous evolution of multiple concentration modulations across multiple length scales, leading to hierarchical morphologies. We show that locally, the concentration must be above a certain threshold value in order to generate distinct hierarchical morphologies in a single domain.

Inhalable particles containing isoniazid and rifabutin as adjunct therapy for safe, efficacious and relapse-free cure of experimental animal tuberculosis in one month.

We investigated the preclinical efficacy and safety/tolerability of biodegradable polymeric particles containing isoniazid (INH) and rifabutin (RFB) dry powder for inhalation (DPI) as an adjunct to oral first-line therapy. Mice and guinea pigs infected with Mycobacterium tuberculosis H37Rv (Mtb) were treated with ∼80 and ∼300 µg of the DPI, respectively, for 3-4 weeks starting 3, 10, and 30 days post-infection. Adjunct combination therapy eliminated culturable Mtb from the lungs and spleens of all but one of 52 animals that received the DPI. Relapse-free cure was not achieved in one mouse that received DPI + oral, human-equivalent doses (HED) of four drugs used in the Directly Observed Treatment, Short Course (DOTS), starting 30 days post-infection. Oral doses (20 mg/Kg/day, each) of INH + RFB reduced Mtb burden from ∼106 to ∼103 colony-forming units. Combining half the oral dose with DPI prevented relapse of infection four weeks after stopping the treatment. The DPI was safe in rodents, guinea pigs, and monkeys at 1, 10, and 100 µg/day doses over 90 days. In conclusion, we show the efficacy and safety/tolerability of the DPI as an adjunct to oral chemotherapy in three different animal models of TB.

Estimation of common location parameter of several heterogeneous exponential populations based on generalized order statistics.

In this article, several independent populations following exponential distribution with common location parameter and unknown and unequal scale parameters are considered. From these populations, several independent samples of generalized order statistics (gos) are drawn. Under the setup of gos, the problem of estimation of common location parameter is discussed and various estimators of common location parameter are derived. The authors obtained maximum likelihood estimator (MLE), modified MLE and uniformly minimum variance unbiased estimator of common location parameter. Furthermore, under scaled-squared error loss function, a general inadmissibility result of invariant estimator is proposed. The derived results are further reduced for upper record values which is a special case of gos. Finally, simulation study and real life example are reported to show the performances of various competing estimators in terms of percentage risk improvement.

Large global variations in measured airborne metal concentrations driven by anthropogenic sources.

Globally consistent measurements of airborne metal concentrations in fine particulate matter (PM2.5) are important for understanding potential health impacts, prioritizing air pollution mitigation strategies, and enabling global chemical transport model development. PM2.5 filter samples (N ~ 800 from 19 locations) collected from a globally distributed surface particulate matter sampling network (SPARTAN) between January 2013 and April 2019 were analyzed for particulate mass and trace metals content. Metal concentrations exhibited pronounced spatial variation, primarily driven by anthropogenic activities. PM2.5 levels of lead, arsenic, chromium, and zinc were significantly enriched at some locations by factors of 100-3000 compared to crustal concentrations. Levels of metals in PM2.5 and PM10 exceeded health guidelines at multiple sites. For example, Dhaka and Kanpur sites exceeded the US National Ambient Air 3-month Quality Standard for lead (150 ng m-3). Kanpur, Hanoi, Beijing and Dhaka sites had annual mean arsenic concentrations that approached or exceeded the World Health Organization's risk level for arsenic (6.6 ng m-3). The high concentrations of several potentially harmful metals in densely populated cites worldwide motivates expanded measurements and analyses.

Mechanical performance of co-deposited immiscible Cu-Ta thin films.

The immiscible alloy Cu-Ta has the potential for enhanced mechanical performance in applications as a functional coating. To establish baseline mechanical properties, four Cu-Ta films were co-sputtered at the temperatures 23, 400, 600, and 800 °C and tested with nanoindentation at strain rates 5 [Formula: see text] 10-3 s-1 to 10 s-1. Each film had a unique microstructure morphology. The hardness and elastic modulus of the four films were insensitive to strain rate changes. Instead, the measured properties were spatially dependent, particularly in the 600 and 800 °C films. In those two films, there is a bimodal deformation behavior due to Cu-agglomeration under protruding grains and planar Ta-rich regions. Increasing the indentation depth revealed shear band suppression which is related to a homogenous distribution of flow stresses for all four microstructure morphologies. Finally, the Cu-Ta hardness appeared to follow a rule-of-mixtures when compared to extrapolated data of Cu and Ta monolithic films.

Transient Transfection of the Respiratory Epithelium with Gamma Interferon for Host-Directed Therapy in Pulmonary Tuberculosis.

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.

A quantitative study of stress fields ahead of a slip band blocked by a grain boundary in unalloyed magnesium.

Stress localization ahead of a slip band blocked by a grain boundary is measured for three different grain boundaries in unalloyed Mg using high-resolution electron backscatter diffraction (HR-EBSD). The results are compared with a theoretical dislocation pile-up model, from which slip system resistance and micro-Hall-Petch coefficients for different grain boundary types are deduced. The results indicate that grain boundary character plays a crucial role in determining micro-Hall-Petch coefficients, which can be used to strengthen classical crystal plasticity constitutive models to make predictions linked to the effect of grain boundary strengthening.

Streptococcus pyogenes purpura fulminans and septic shock: A case highlighting the ethical considerations of high-risk extracorporeal membrane oxygenation.

Separately, refractory septic shock and purpura fulminans have very poor outcomes. The ethics involved in offering extracorporeal membrane oxygenation to very high-risk patients is complex. We report a novel case of refractory shock requiring veno-arterial extracorporeal membrane oxygenation and continuous renal replacement therapy due to Streptococcus pyogenes bacteremia with purpura fulminans to highlight the ethical challenges in offering extracorporeal membrane oxygenation to a patient with such a poor likelihood of survival.

Carotid Flow as a Surrogate for Cardiac Output Measurement in Hemodynamically Stable Participants.

OBJECTIVE: Evaluation of common carotid artery (CCA) blood flow can provide valuable information regarding the hemodynamic status of a patient. Utilizing ultrasound, we aimed to evaluate the correlation between cardiac output and different hemodynamic parameters in the CCA, namely systolic carotid flow (SCF), corrected flow time (CFT), and total carotid flow (TCF). METHODS: We studied a pilot sample of 20 healthy volunteers. Hemodynamic parameters were collected in the right CCA and the heart at rest (baseline), 1-leg compression, 2-leg compression, and passive leg raise. Nonparametric Spearman correlation was calculated using STATA 13 software. RESULTS: This study demonstrated the feasibility and safety of the leg compression testing as a hemodynamic maneuver to simulate volume depletion status. We demonstrated a direct correlation between cardiac output and SCF of 0.67 with a P value < 0.001. Interestingly, TCF calculated based on volume-time integral (VTI) in the carotid artery showed positive correlation of only 0.41, with P < 0.06, and it did not reach statistical significance. We also found a positive correlation between CFT and cardiac output at baseline 0.57, with P < 0.001. CONCLUSION: Variations in cardiac preload and the subsequent alterations in cardiac output were directly translatable into variations in the carotid blood flow. This supports the potential for using carotid flow as a surrogate for cardiac output. The most promising parameters were SCF, CFT, and carotid systolic VTI. Further work is needed to validate these correlations and utilize these acquired carotid parameters to guide fluid management and predict fluid responsiveness.

Opioid-induced toxic leukoencephalopathy: A case report and review of the literature.

IMPORTANCE: Reports of toxic leukoencephalopathy (TLE) due to opioids have been extensively documented within the adult literature. There is a paucity of literature with respect to the incidence, complications, and outcomes of TLE in the pediatric population. OBJECTIVE: To describe a rare complication of opioid ingestion in the pediatric population and serve as the first large review of published cases of opioid-induced leukoencephalopathy. Thirteen case reports with varying treatments are herein reviewed in addition to our own case. The range of treatment modalities, morbidity and mortality are broad and outcomes secondary to supportive care versus neurosurgical intervention is explored. EVIDENCE REVIEW: All cases of pediatric opioid-induced toxic leukoencephalopathy published on pubmed and google scholar were included in this review. FINDINGS: We report the case of a 4-year old male surgically treated for acute oxycodone-induced TLE who initially presented with Glascow Coma Scale of 4 and a comatose state for weeks. Over the next several months he recovered with spasticity of all extremities, oral aversion, substantial vision loss, and the ability to speak in short sentences. In addition, we found thirteen other reported cases of opioid-induced leukoencephalopathy reported in the literature. The treatment approaches described range from supportive care alone, to invasive neurosurgical interventions including placement of extraventricular drains, removal of hemorrhagic tissue, and craniectomy. The outcomes of patients with opioid-induced leukoencephalopathy is also variable. Reports demonstrate a range of outcomes that include patients who died to those with no residual neurologic deficits. CONCLUSIONS: This review of reported pediatric cases of opioid-induced leukoencephalopathy highlights the importance of early neurosurgical intervention for prevention of devastating outcomes.

The Use of an Autologous Cell Harvesting and Processing Device to Decrease Surgical Procedures and Expedite Healing in Two Pediatric Burn Patients.

INTRODUCTION: Autologous cell harvesting and processing devices are designed to facilitate the harvesting of cells using enzymatic and physical disruption techniques to immediately apply non-cultured autologous cell suspension (ACS) to the wound area. OBJECTIVE: This case report evaluates clinical outcomes following application of cellular suspension with split-thickness skin grafts (STSGs) as an adjunct for definitive closure of burn injuries and donor sites in 2 pediatric patients. MATERIALS AND METHODS: The cases were performed under a humanitarian use protocol following institutional review board approval at St. Christopher's Hospital for Children (Philadelphia, PA). RESULTS: The first patient was a 4-year-old girl with partial- and full-thickness (32% total body surface area) burn injuries of her head, trunk, flank, arms, thighs, and feet. The patient was discharged 19 days following ACS treatment. The second patient was an 18-month-old girl with partial- and full-thickness (21% total body surface area) burns involving the bilateral lower extremities. She was discharged 22 days after ACS treatment with widely meshed autograft. Neither patient required additional surgical interventions. All treatment and donor areas for both patients remained uninfected and neither patient experienced any unexpected treatment-related adverse events. CONCLUSIONS: These cases are the first of their kind reported in the pediatric population and suggest ACS in conjunction with STSGs can help decrease surgical procedures and expedite healing in pediatric patients with large surface burns.