Respiratory Challenge Research Articles

Validating layer-specific VASO across species

Cerebral blood volume (CBV) has been shown to be a robust and important physiological parameter for quantitative interpretation of functional (f)MRI, capable of delivering highly localized mapping of neural activity. Indeed, with recent advances in ultra-high-field (≥7T) MRI hardware and associated sequence libraries, it has become possible to capture non-invasive CBV weighted fMRI signals across cortical layers. One of the most widely used approaches to achieve this (in humans) is through vascular-space-occupancy (VASO) fMRI. Unfortunately, the exact contrast mechanisms of layer-dependent VASO fMRI have not been validated for human fMRI and thus interpretation of such data is confounded. Here we validate the signal source of layer-dependent SS-SI VASO fMRI using multi-modal imaging in a rat model in response to neuronal activation (somatosensory cortex) and respiratory challenge (hypercapnia). In particular VASO derived CBV measures are directly compared to concurrent measures of total haemoglobin changes from high resolution intrinsic optical imaging spectroscopy (OIS). Quantified cortical layer profiling is demonstrated to be in agreement between VASO and contrast enhanced fMRI (using monocrystalline iron oxide nanoparticles, MION). Responses show high spatial localisation to layers of cortical processing independent of confounding large draining veins which can hamper BOLD fMRI studies, (depending on slice positioning). Thus, a cross species comparison is enabled using VASO as a common measure. We find increased VASO based CBV reactivity (3.1 ± 1.2 fold increase) in humans compared to rats. Together, our findings confirm that the VASO contrast is indeed a reliable estimate of layer-specific CBV changes. This validation study increases the neuronal interpretability of human layer-dependent VASO fMRI as an appropriate method in neuroscience application studies, in which the presence of large draining intracortical and pial veins limits neuroscientific inference with BOLD fMRI.

Ampakines enhance phrenic nerve output in a ZFN rat model of Pompe Disease

Pompe disease (PD) is caused by mutations in the gene encoding acid-α-glucosidase (GAA), an enzyme that degrades lysosomal glycogen. The resultant glycogen accumulation is associated with neurodegeneration, cardiac deficits, and impaired phrenic motoneuron and diaphragm activation. Cardiorespiratory failure is the leading cause of death in early-onset PD patients. We previously reported that a drug which acts as a positive allosteric modulator of AMPA receptors, ampakine CX717, could stimulate phrenic motor output and increase minute ventilation in a mouse model of Pompe disease (Gaa-/-). We recently created a new rat model of PD that better recapitulates the clinical phenotype of early onset PD, called the Zinc Finger Nuclease (ZFN) KO model. Phrenic nerve recordings were performed on urethane anesthetized ZFN KO rats of both sexes, and age-matched wild type (WT) controls during a baseline normoxic period, followed by a brief exposure to hypoxia (PaO2 ~ 30mmHg) and a maximal respiratory challenge with combined hypoxia-hypercapnia. Recordings were done before and after treatment with ampakine CX717 (15mg/kg) or its vehicle control (2-Hydroxypropyl)-β-cyclodextrin. Our goal was to build upon the prior data from the Gaa-/-mouse, by using the ZFN KO model to test the hypothesis that in addition to stimulating basal or “eupneic” phrenic output, ampakine CX717 could also increase the ability to respond to acute respiratory challenge. We also aimed to determine if the extent of phrenic motor deficits were different between male and female ZFN KO rats. The data confirm that both male and female ZFN KO rats exhibit cardiac irregularities and phrenic motor deficits when compared to WT controls. However, a sex difference was apparent as male ZFN KO rats exhibit greater phrenic motor deficits and blunted responses to gas challenges compared to female ZFN KO rats. Ampakine treatment enhanced phrenic output and improved the ability to respond to respiratory challenge in both sexes of the ZNF rat model of PD. We conclude that 1) sex differences are present in the phrenic motor phenotype of ZFN KO rats, 2) the ability of low dose CX717to stimulate phrenic motor output in PD is reproducible across two species, and 3) low dose CX717 can enhance the ability to respond to respiratory challenge in PD rats These data suggest that ampakines may serve as a viable adjunctive therapy for respiratory insufficiency in PD patients.

The Long‐Term Impact of Ovariectomy on Ventilation and Expression of Phrenic Long‐Term Facilitation in Female Rats

Estrogen is necessary for the expression of phrenic long-term facilitation (pLTF) in female rats. pLTF, a well-studied model of respiratory neuroplasticity, is characterized by a progressive increase in phrenic nerve amplitude following acute intermittent hypoxia (AIH). Gonadally-intact, adult female rats only develop pLTF in stages of the estrous cycle with high levels of circulating estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating estrogen in females, also prevents AIH-induced pLTF and estrogen supplementation is sufficient to restore pLTF in OVX rats, supporting a key role for estrogen in the development of pLTF. Respiratory neuroplasticity studies using OVX female rats are generally completed ~7-10 days post-OVX; a timeframe sufficient for surgery recovery and for circulating estrogen levels to significantly decline. However, few studies have explored the long-term impact of OVX on respiratory function or the development of respiratory neuroplasticity. Here, we examined ventilation in awake, freely behaving rats and the development of AIH-induced pLTF in adult female rats 12 weeks-post OVX (or sham control surgeries) and compared them to female rats 2 weeks-post OVX. Our hypothesis was that chronic OVX would have minimal long-term impact on ventilation but would continue to impair development of AIH-induced pLTF. Whole body plethysmography was used to measure baseline breathing (20.9% O2, N2 balance) and ventilatory responses to strong chemoreceptor stimulation (10% O2, 5% CO2, N2 balance). Respiratory measures including frequency, tidal volume, minute ventilation, and respiratory neural drive were measured and compared between groups. Following plethysmography, phrenic nerve recordings were used to quantify pLTF. Results indicate that chronic OVX had little impact on baseline breathing function and rats in all experimental groups responded appropriately to respiratory challenge during plethysmography. However, 12 week OVX rats showed a significant increase in tidal volume relative to baseline during respiratory challenge (p≤0.001), different from other groups. This increase in tidal volume production was also reflected in the calculation of tidal volume/inspiratory time, a measure of relative respiratory neural drive (p≤0.01) but not in minute ventilation. OVX had sustained effects on development of respiratory plasticity, as only rats in the 12 week sham control group displayed pLTF, indicated by significantly increased phrenic nerve amplitude 60-min following AIH (p≤0.001). This increase in amplitude was higher than both 12 week and 2 week OVX groups (p≤0.01). ELISA assays also showed a significant correlation between circulating estrogen levels and magnitude of pLTF (p= 0.039) In summary, OVX appears to limit the expression of respiratory neuroplasticity in female rats for up to 12 weeks. Since no significant changes in circulating estrogen are observed in OVX rats over that time, these data are consistent with our prior work indicating that estrogen plays a key role in development of respiratory neuroplasticity.

Low Dose Ampakine Stimulates Diaphragm Activity and Increases Tidal Volume following Cervical Spinal Cord Injury in Non‐Anesthetized Freely Behaving Rats

Sabhya Rana1, 2, 3, Michael D. Sunshine1, 2, 3, David D. Fuller1, 2, 3 1Department of Physical Therapy, 2Center for Respiratory Research and Rehabilitation, 3McKnight Brain Institute, University of Florida, Gainesville, FL Ampakines are allosteric modulators of AMPA receptor channel kinetics, and can stimulate breathing during hypoventilation such as following opioid overdose or in neuromuscular disorders. Our laboratory has shown that acute treatment with an ampakine, CX717 can increase inspiratory phrenic motor output in rat models of incomplete cervical spinal cord injury (SCI), when studied under anesthesia. The next step in the translational pathway to is to determine if the drugs are safe and effective in the awake animal. The goal of the current work was to evaluate the effect of two low impact ampakines (CX717 and CX1739) on diaphragm muscle activation and breathing in freely moving non-anesthetized rats following SCI. Ventilation and metabolic rate (VCO2) were measured using whole body plethysmography and diaphragm EMG was assessed using in-dwelling muscle electrodes. At 4 and 14 days following C2 spinal hemisection injury (C2Hx), rats were given a single intravenous bolus of CX717 (5 mg/kg, n=8), CX1739 (5 mg/kg, n=8) or vehicle (2-hydroxypropyl-beta-cyclodextrin; HPCD; n=8). At the 4-day time point, infusion of CX717 or CX1739 increased peak diaphragm EMG (recorded ipsilateral to C2Hx) by 40-60% compared to vehicle. However, only the CX1739 group showed an accompanying increase in tidal volume (20%). During an acute respiratory challenge with hypoxia-hypercapnia (10% O2, 7% CO2), both the CX717 and CX1739 groups showed increased diaphragm EMG compared to vehicle (65-75%); the CX1739 group showed a larger increase in tidal volume. At 14 days post injury, CX717 infusion increased the peak inspiratory ipsilateral diaphragm EMG activity (~30%) and tidal volume (~20%) during baseline breathing. In contrast, CX1739 was less effective at 14 days and produced an increase in ipsilateral diaphragm EMG activity of ~10% with no change in tidal volume. During the hypoxia-hypercapnia challenge, both the CX1739 and CX717 groups showed increased ipsi-lesional diaphragm EMG (25-35%) but no increase in tidal volume as compared to vehicle group. Neither ampakine had an impact on respiratory rate at either time point, suggesting a spinal (vs. medullary) mechanism of action. Additionally, no adverse off-target effects (e.g., changes in heart rate or hypoactive state) were apparent. Improved respiratory muscle activation is often a desirable therapeutic outcome after incomplete cervical SCI. Our data indicate that low dose, low impact ampakine treatment can increase diaphragm muscle output after cervical SCI, and therefore may provide a pharmacologic strategy that could be useful in the context of respiratory rehabilitation. The divergent response to CX717 vs. CX1739 at 4 vs. 14 days post-injury merits further study; one possibility is that ongoing neuroplastic changes in AMPA receptor expression after SCI may differentially influence the efficacy of different ampakines.

Time course of ventilatory failure in a novel rat model of Pompe Disease

Pompe disease (PD) is caused by mutations in the gene encoding the enzyme acid-α-glucosidase (GAA). Since GAA acts to degrade lysosomal glycogen, reduced or loss of enzyme activity leads to glycogen accumulation and neuromuscular impairments. Respiratory dysfunction and ventilator-dependence is common in both early- and late-onset PD. Our group has created a rat model of PD using Zinc Finger Nuclease (ZFN) technology. The goal of the current study was to characterize ventilation and metabolism in unrestrained ZFN Pompe rats using whole body plethysmography. Data were collected during a normoxic baseline period, acute exposures to hypoxia (FiO2=10.5%), hypercapnia (FiCO2=7%), and combined hypercapnic-hypoxic challenge intended to produce a maximal ventilatory drive. ZFN Pompe rats and age-matched, wild type (WT) controls were studied at age 3, 6, and 10-11 months to assess ventilation throughout disease progression. Both male and female 3 month old ZFN Pompe rats hyperventilated (greater VE/VCO2) due to greater tidal volume compared to WT rats. However, as the ZFN Pompe rats aged, the ventilatory phenotype changed such that by 10-11 months they showed significant hypoventilation as well as a blunted ability to increase ventilation in response to respiratory challenge relative to WT controls. Histological data from these rats, as well as prior data from the Gaa-/- mouse model of PD, show that respiratory neuromuscular pathology is present at a very young age. Accordingly, the current ventilatory data suggest that animals with PD initially increase breathing in response to the onset of pathology, possibly representing a period of “overcompensation”. However, this is followed by respiratory failure as the respiratory neuromuscular pathology progresses towards disease end-stage.

Immunogenicity and protectivity of the peptide candidate vaccine against SARS-CoV-2

Background: In 2020, the pandemic caused by novel coronavirus infection has become one of the most critical global health challenges during the past century. The lack of a vaccine, as the most effective way to control the novel infection, has prompted the development of a large number of preventive products by the scientific community. We have developed a candidate vaccine (EpiVacCorona) against novel coronavirus infection caused by SARS-CoV-2 that is based on chemically synthesized peptides conjugated to a carrier protein and adsorbed on aluminum hydroxide and studied the specific activity of the developed vaccine.
 Aims: Study of the immunogenicity and protectivity of the peptide candidate vaccine EpiVacCorona.
 Materials and methods: the work was performed using standard molecular biological, virological and histological methods.
 Results: It was demonstrated that EpiVacCorona, when administered twice, spaced 14 days apart, to hamsters, ferrets, and non-human primates (African green monkeys, rhesus macaques) at a dose of 260 g, which is equal to one inoculation dose for humans, induces virus-specific antibodies in 100% of the animals. Experiments in hamsters showed this vaccine to be associated with the dose-dependent immunogenicity. The vaccine was shown to accelerate the elimination of the virus from the upper respiratory tract in ferrets and prevent the development of pneumonia in hamsters and non-human primates following a respiratory challenge with novel coronavirus.
 Conclusions: The results of a preclinical specific activity study indicate that the use of EpiVacCorona has the potential for human vaccination.

Zinc Source and Concentration Altered Physiological Responses of Beef Heifers during a Combined Viral-Bacterial Respiratory Challenge.

Simple SummaryBovine respiratory disease is one of the greatest health challenges cattle producers face and is most commonly treated with antibiotics. With the current push to reduce the use of antibiotics in livestock production, producers are looking at non-pharmaceutical alternatives such as nutritional supplements. This study aimed to determine if different forms of zinc supplementation could reduce some of the negative health effects associated with bovine respiratory disease. Overall, cattle supplemented with ZinMet (zinc methionine/organic zinc) responded better during the disease as evidenced by blood parameters, decreased lesion severity, and decreased fever. Conversely, cattle fed a large dose of zinc sulfate (inorganic zinc) displayed a higher fever and blood parameters that indicated a greater sickness response. Findings from this study suggest that the type and amount of zinc fed to cattle may influence their response to bovine respiratory disease.To determine the effects of zinc supplementation on the immune response to a combined viral-bacterial respiratory disease challenge, thirty-two beef heifers (255 ± 15 kg) were subjected to a 30-d period of Zn depletion, then randomly assigned to one of three treatment diets fed for 30 d before the challenge: (1) supplementation with 100 mg of Zn from Zn sulfate/kg of DM (Zn100), (2) supplementation with 200 mg of Zn from Zn sulfate/kg of DM (Zn200), and (3) supplementation with 80 mg of Zn/kg of DM from zinc methionine and 20 mg of Zn from Zn sulfate/kg of DM (ZinMet). After the 30-d supplementation period, all heifers were fitted with indwelling vaginal temperature (VT) devices and intra-nasally challenged with 1 × 108 PFU bovine herpesvirus-1 on d -3, and then allowed to rest in outdoor pens for 3 d. On d 0, each heifer was challenged intra-tracheally with an average dose of 2.38 × 107 CFU Mannheimia haemolytica (MH), fitted with an indwelling jugular catheter, and then moved into individual stalls in an environmentally-controlled enclosed barn. Whole blood samples were collected at 1-h (serum) and 2-h (complete blood counts) intervals from 0 to 8 h, and at 12, 24, 36, 48, 60, 72, 168, and 360 h relative to MH challenge. Data were analyzed using the MIXED procedure of SAS specific for repeated measures with fixed effects of treatment, time, and their interaction. There was a treatment effect (p < 0.01) for VT such that Zn200 heifers had greater VT than Zn100 and ZinMet heifers. There was a trend (p = 0.10) for a serum cortisol treatment effect with Zn100 heifers having greater cortisol than ZinMet heifers. Total leukocytes and lymphocytes were greater (p ≤ 0.01) in Zn100 heifers than Zn200 and ZinMet heifers, whereas monocytes were less (p = 0.05) in ZinMet heifers than Zn100 and Zn200 heifers. Concentrations of IL-6 were greater (p = 0.02) in ZinMet heifers than Zn100 and Zn200 heifers. Concentrations of IFN-γ were greater in Zn200 heifers than ZinMet heifers at 0 h, and Zn100 heifers from 0 to 12 h post-MH challenge (treatment x time p = 0.02). Serum haptoglobin was not affected by treatment or treatment x time (p ≥ 0.36) but increased over time (p < 0.01) in all groups. There was a trend (p = 0.11) for ZinMet heifers to have less severe nasal lesion scores than Zn100 heifers. The observed differential physiological responses in this study indicate that zinc source and concentration may alter the response to a bovine respiratory challenge in heifers.

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this study was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches

Induction of memory CD8 T cells residing in peripheral tissues is of interest for T cell-based vaccines as these cells are located at mucosal and barrier sites and can immediately exert effector functions, thus providing protection in case of local pathogen encounter. Different memory CD8 T cell subsets patrol peripheral tissues, but it is unclear which subset is superior in providing protection upon secondary infections. We used influenza virus to induce predominantly tissue resident memory T cells or cytomegalovirus to elicit a large pool of effector-like memory cells in the lungs and determined their early protective capacity and mechanism of reactivation. Both memory CD8 T cell pools have unique characteristics with respect to their phenotype, localization, and maintenance. However, these distinct features do not translate into different capacities to control a respiratory vaccinia virus challenge in an antigen-specific manner, although differential activation mechanisms are utilized. While influenza-induced memory CD8 T cells respond to antigen by local proliferation, MCMV-induced memory CD8 T cells relocate from the vasculature into the tissue in an antigen-independent and partially chemokine-driven manner. Together these results bear relevance for the development of vaccines aimed at eliciting a protective memory CD8 T cell pool at mucosal sites.

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Epicutaneous immunization with modified vaccinia Ankara viral vectors generates superior T cell immunity against a respiratory viral challenge

Modified Vaccinia Ankara (MVA) was recently approved as a smallpox vaccine. Variola is transmitted by respiratory droplets and MVA immunization by skin scarification (s.s.) protected mice far more effectively against lethal respiratory challenge with vaccinia virus (VACV) than any other route of delivery, and at lower doses. Comparisons of s.s. with intradermal, subcutaneous, or intramuscular routes showed that MVAOVA s.s.-generated T cells were both more abundant and transcriptionally unique. MVAOVA s.s. produced greater numbers of lung Ova-specific CD8+ TRM and was superior in protecting mice against lethal VACVOVA respiratory challenge. Nearly as many lung TRM were generated with MVAOVA s.s. immunization compared to intra-tracheal immunization with MVAOVA and both routes vaccination protected mice against lethal pulmonary challenge with VACVOVA. Strikingly, MVAOVA s.s.-generated effector T cells exhibited overlapping gene transcriptional profiles to those generated via intra-tracheal immunization. Overall, our data suggest that heterologous MVA vectors immunized via s.s. are uniquely well-suited as vaccine vectors for respiratory pathogens, which may be relevant to COVID-19. In addition, MVA delivered via s.s. could represent a more effective dose-sparing smallpox vaccine.

64 Immunological, physiological, and behavioral responses to an experimentally-induced respiratory disease challenge in growing steers

Abstract Immunologic, physiologic, and behavioral responses to a combined viral-bacterial respiratory challenge were explored in beef steers (initial BW 293 kg). Steers (n = 24) were inoculated intranasally with bovine herpes virus-1 (2×108 PFU) and intratracheally with Mannheimia haemolytica (MH, 2.15×1010 CFU) on days -3 and 0, respectively, (n = 16; VB), or similarly inoculated with phosphate-buffered saline (n = 8; PBS). Venous and arterial blood were collected on -3, -1, 0, 2, 3, 5, 7, 10, and 14 relative to MH challenge for CBC, haptoglobin, and arterial blood oxygen saturation analysis. Continuously recorded variables included rumen temperature, activity, rumination, DMI, and feeding behavior. Data were analyzed with a repeated-measures mixed model (SAS 9.4) with fixed effects of day, inoculation, and the interaction. Bunk visit frequency and DMI were reduced (P &amp;lt; 0.01) in VB steers throughout the 14 d post-MH period compared to PBS steers. Rumination (days 1, 7) and activity (days 1, 2, 4–8, and 11–13) were reduced (P &amp;lt; 0.03) in VB steers vs PBS steers. Rumen temperature was elevated (P &amp;lt; 0.04) in VB steers until day 6 post MH inoculation. Neutrophil concentrations (days 2 and 3), platelets (days 7–14), fibrinogen (days 2–10), and haptoglobin (days 2–7) were elevated (P &amp;lt; 0.05) in VB vs PBS steers. Hematocrit was depressed (P &amp;lt; 0.05) in VB steers on days 3–10. The VB steers had decreased (P &amp;lt; 0.05) arterial SO2% and pO2 than PBS steers; however, the inoculation × day interaction was not significant. Results indicate that the experimental VB challenge substantially altered rumen temperature, DMI, feeding behavior, rumination and immunological response as expected. Arterial sO2% and pO2 concentrations were reduced minimally by VB challenge, indicating challenges in using blood gas to detect BRD in beef cattle.

Safety and Antiviral Effects of Nebulized PC786 in a Respiratory Syncytial Virus Challenge Study.

BackgroundPC786 is a nebulized nonnucleoside respiratory syncytial virus (RSV) polymerase inhibitor designed to treat RSV, which replicates in the superficial layer of epithelial cells lining the airways.MethodsFifty-six healthy volunteers inoculated with RSV-A (Memphis 37b) were randomly dosed with either nebulized PC786 (5 mg) or placebo, twice daily for 5 days, from either 12 hours after confirmation of RSV infection or 6 days after virus inoculation. Viral load (VL), disease severity, pharmacokinetics, and safety were assessed until discharge. RSV infection was confirmed by reverse-transcription quantitative polymerase chain reaction with any positive value (intention-to-treat infected [ITT-I] population) or RSV RNA ≥1 log10 plaque-forming unit equivalents (PFUe)/mL (specific intention-to-treat infection [ITT-IS] population) in nasal wash samples.ResultsIn the ITT-I population, the mean VL area under the curve (AUC) was lower in the PC786 group than the placebo group (274.1 vs 406.6 log10 PFUe/mL × hour; P = .0359). PC786 showed a trend toward reduction of symptom score and mucous weight. In ITT-IS (post hoc analysis), the latter was statistically significant as well as VL AUC (P = .0126). PC786 showed an early time to maximum plasma concentration, limited systemic exposure, and long half-life and consequently a 2-fold accumulation over the 5-day dosing period. PC786 was well tolerated.ConclusionsNebulized PC786 demonstrated a significant antiviral effect against RSV, warranting further clinical study.Clinical Trials RegistrationClinicalTrials.gov: NCT03382431; EudraCT: 2017-002563-18.

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to Streptococcus pneumoniae.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.

Permanent diaphragmatic deficits and spontaneous respiratory plasticity in a mouse model of incomplete cervical spinal cord injury

High spinal cord injuries (SCI) lead to permanent respiratory insufficiency, and the search for new therapeutics to restore this function is essential. To date, the most documented preclinical model for high SCI is the rat cervical C2 hemisection. However, molecular studies with this SCI model are limited due to the poor availability of genetically modified specimens. The aim of this work was to evaluate the pathophysiology of respiratory activity following a cervical C2 injury at different times post-injury in a C57BL/6 mouse model. No significant spontaneous recovery of diaphragmatic activity was observed up to 30 days post-injury in eupneic condition. However, during a respiratory challenge, i.e. mild asphyxia, a partial restoration of the injured diaphragm was observed at 7 days post-injury, corresponding to the crossed phrenic phenomenon. Interestingly, the diaphragmatic recording between 2 respiratory bursts on the injured side showed an amplitude increase between 1–7 days post-injury, reflecting a change in phrenic motoneuronal excitability. This increase in inter-burst excitability returned to pre-injured values when the crossed phrenic phenomenon started to be effective at 7 days post-injury. Taken together, these results demonstrate the ability of the mouse respiratory system to express long-lasting plasticity following a C2 cervical hemisection and genetically modified animals can be used to study the pathophysiological effects on these plasticity phenomena.

Induction of protection in mice against a respiratory challenge by a vaccine formulated with exosomes isolated from Chlamydia muridarum infected cells

The goal of this study was to determine if exosomes, isolated from Chlamydia muridarum infected HeLa cells (C. muridarum-exosomes), induce protective immune responses in mice following vaccination using CpG plus Montanide as adjuvants. Exosomes, collected from uninfected HeLa cells and PBS, formulated with the same adjuvants, were used as negative controls. Mass spectrometry analyses identified 113 C. muridarum proteins in the C. muridarum-exosome preparation including the major outer membrane protein and the polymorphic membrane proteins. Vaccination with C. muridarum-exosomes elicited robust humoral and cell-mediated immune responses to C. muridarum elementary bodies. Following vaccination, mice were challenged intranasally with C. muridarum. Compared to the negative controls, mice immunized with C. muridarum-exosomes were significantly protected as measured by changes in body weight, lungs’ weight, and number of inclusion forming units recovered from lungs. This is the first report, of a vaccine formulated with Chlamydia exosomes, shown to elicit protection against a challenge.

Respiratory pathology in the Optn-/- mouse model of Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder that results in death due to respiratory failure. Many genetic defects are associated with ALS; one such defect is a mutation in the gene encoding optineurin (OPTN). Using an optineurin null mouse (Optn-/-), we sought to characterize the impact of optineurin deficiency on respiratory neurodegeneration. Respiratory function was assessed at 6 and 12 mo of age using whole body plethysmography at baseline during normoxia (FiO2: 0.21; N2 balance) and during a respiratory challenge with hypoxia and hypercapnia (FiCO2: 0.07, FiO2: 0.10; N2 balance). Histological analyses to assess motor neuron viability and respiratory nerve integrity were performed in the medulla, cervical spinal cord, hypoglossal nerve, and phrenic nerve. Minute ventilation, peak inspiratory flow, and peak expiratory flow are significantly reduced during a respiratory challenge in 6 mo Optn-/-mice. By 12 mo, tidal volume is also significantly reduced in Optn-/- mice. Furthermore, 12mo Optn-/- mice exhibit hypoglossal motor neuron loss, phrenic and hypoglossal dysmyelination, and accumulated mitochondria in the hypoglossal nerve axons. Overall, these data indicate that Optn-/- mice display neurodegenerative respiratory dysfunction and are a useful model to study the impact of novel therapies on respiratory function for optineurin-deficient ALS patients.

Cerebrovascular Reactivity After Sport Concussion: From Acute Injury to 1 Year After Medical Clearance.

Neuroimaging has identified significant disturbances in cerebrovascular reactivity (CVR) in the early symptomatic phase of sport-related concussion. However, less is known about how whole-brain alterations in CVR evolve after concussion and whether they remain present beyond medical clearance to return to play (RTP). In the present study, CVR was evaluated using blood-oxygenation-level-dependent functional magnetic resonance imaging (BOLD fMRI) during a respiratory challenge. Imaging data were collected for 110 university-level athletes, including 39 concussed athletes and 71 athletic controls. The concussed athletes were imaged at the acute phase of injury (1–7 days post-injury), the subacute phase (8-14 days post-injury), medical clearance to RTP, 1 month post-RTP, and 1 year post-RTP. Enhanced negative BOLD response to controlled breathing was seen at acute injury, with attenuation of the effect mainly occurring by 1 year post-RTP. Secondary analyses showed that greater symptom severity and prolonged recovery were associated with enhanced BOLD response in the acute phase of injury, but a more attenuated BOLD response in the subacute phase. This study provides novel information characterizing the CVR response after concussion and shows CVR to be a sensitive technique for evaluating long-term brain recovery.

Protection against a chlamydial respiratory challenge by a chimeric vaccine formulated with the Chlamydia muridarum major outer membrane protein variable domains using the Neisseria lactamica porin B as a scaffold

Chlamydia trachomatis is the most frequently detected sexually transmitted bacterial pathogen in the world. Attempts to control these infections with screening programs and antibiotics have failed and, therefore, a vaccine is the best approach to control this epidemic. The Chlamydia major outer membrane protein (MOMP) is the most protective subunit vaccine so far tested. Protection induced by MOMP is, in part, dependent on its tertiary structure. We have previously described new recombinant antigens composed of the Neisseria lactamica PorB engineered to express the variable domains (VD) from Chlamydia muridarum MOMP. Here we tested antigens containing each individual MOMP VD and different VD combinations. Following immunization, mice were challenged intranasally with C. muridarum. Our results show that three constructs, PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4, elicited high serum IgG titers in vivo, significant IFN-γ levels upon T cells re-stimulation in vitro, and evidence of protective immunity in vivo. PorB/VD1–3, PorB/VD1–4, and PorB/VD1–2–4 immunized mice lost less body weight, had lighter lungs, and decreased numbers of inclusion forming units (IFUs) in lungs than other PorB/VD construct tested and mock PBS-immunized mice. These results suggest that this approach may be a promising alternative to the use of MOMP in a Chlamydia vaccine.

Subchronic inhalation of Aspergillus versicolor conidia leads to sustained innate immune responses and is associated with irreversible remodeling of pulmonary arterial tissue following recovery

Abstract Aspergillus versicolor is an environmentally ubiquitous fungal species that is particularly abundant in environments with water infiltration. Respiration of fungal products, including conidia, hyphal fragments, mycotoxins, and other secondary metabolites may cause adverse pulmonary health effects including asthma and allergy. In the present study, pulmonary immune responses in B6C3F1 mice were examined following 1, 2, 4, 8, and 13 weeks of repeated respiratory challenge to 3×105A. versicolor conidia. Increased alveolar macrophages, inflammatory monocytes, neutrophils, and eosinophils, which peaked between 4 and 8 weeks, were detected in the lungs of mice exposed to viable conidia. Type 2 innate lymphoid cells (ILC2s) peaked between 8 and 13 weeks compared to total T cells which increased between 2 and 8 weeks and decreased at 13 weeks. Protein concentrations of the Th2 cytokines IL4, IL5, and IL13 were highest in bronchoaveolar lavage fluid of exposed mice following 4 weeks and exhibited a time-dependent reduction at 8 and 13 weeks. Inhalation of conidia for 13 weeks led to goblet cell hyperplasia, vascular wall thickening, and perivascular inflammation. Finally, mixed perivascular inflammation and smooth muscle hypertrophy were sustained at 4 weeks post-exposure. These data indicate that exposure to A. versicolor elicits a Th2-biased immune response that is driven by innate cell populations and is associated with the induction of remodeling of the pulmonary vasculature.