Aerosol Delivery of Palivizumab in a Neonatal Lamb Model of Respiratory Syncytial Virus Infection.

(1) Background: Palivizumab has been an approved preventative monoclonal antibody for respiratory syncytial virus (RSV) infection for over two decades. However, due to its high cost and requirement for multiple intramuscular injections, its use has been limited mostly to high-income countries. Following our previous study showing the successful lung deposition of aerosolised palivizumab in lambs, this current study evaluated the "proof-of-principle" effect of aerosolised palivizumab delivered as a therapeutic to neonatal lambs following RSV infection. (2) Methods: Neonatal lambs were intranasally inoculated with RSV-A2 on day 0 (day 3 post-birth) and treated with aerosolised palivizumab 3 days later (day 3 post-inoculation). Clinical symptoms, RSV viral load and inflammatory response were measured post-inoculation. (3) Results: Aerosolised therapeutic delivery of palivizumab did not reduce RSV viral loads in the nasopharynx nor the bronchoalveolar lavage fluid, but resulted in a modest reduction in inflammatory response at day 6 post-inoculation compared with untreated lambs. (4) Conclusions: This proof-of-principle study shows some evidence of aerosolised palivizumab reducing RSV inflammation, but further studies using optimized protocols are needed in order to validate these findings.

Single-dose pharmacokinetics and lung function of nebulized niclosamide ethanolamine in sheep.

PURPOSE: Niclosamide is approved as an oral anthelminthic, but its low oral bioavailability hinders its medical use requiring high drug exposure outside the gastrointestinal tract. An optimized solution of niclosamide for nebulization and intranasal administration using the ethanolamine salt has been developed and tested in a Phase 1 trial. In this study we investigate the pulmonary exposure of niclosamide following administration via intravenous injection, oral administration or nebulization. METHODS: We characterized the plasma and pulmonary pharmacokinetics of three ascending doses of nebulized niclosamide in sheep, compare it to intravenous niclosamide for compartmental PK modelling, and to the human equivalent approved 2 g oral dose to investigate in the pulmonary exposure of different niclosamide delivery routes. Following a single-dose administration to five sheep, niclosamide concentrations were determined in plasma and epithelial lining fluid (ELF). Non-compartmental and compartmental modeling was used to characterize pharmacokinetic profiles. Lung function tests were performed in all dose groups. RESULTS: Administration of all niclosamide doses were well tolerated with no adverse changes in lung function tests. Plasma pharmacokinetics of nebulized niclosamide behaved dose-linear and was described by a 3-compartmental model estimating an absolute bioavailability of 86%. ELF peak concentration and area under the curve was 578 times and 71 times higher with nebulization of niclosamide relative to administration of oral niclosamide. CONCLUSIONS: Single local pulmonary administration of niclosamide via nebulization was well tolerated in sheep and resulted in substantially higher peak ELF concentration compared to the human equivalent oral 2 g dose.

Liposomes are Poorly Absorbed via Lung Lymph After Inhaled Administration in Sheep.

Enhancing the delivery of therapeutic agents to the lung lymph, including drugs, transfection agents, vaccine antigens and vectors, has the potential to significantly improve the treatment and prevention of a range of lung-related illnesses. One way in which lymphatic delivery can be optimized is via the use of nanomaterial-based carriers, such as liposomes. After inhaled delivery however, there is conflicting information in the literature regarding whether nanomaterials can sufficiently access the lung lymphatics to have a therapeutic benefit, in large part due to a lack of reliable quantitative pharmacokinetic data. The aim of this work was to quantitatively evaluate the pulmonary lymphatic pharmacokinetics of a model nanomaterial-based drug delivery system (HSPC liposomes) in caudal mediastinal lymph duct cannulated sheep after nebulized administration to the lungs. Liposomes were labelled with 3H-phosphatidylcholine to facilitate evaluation of pharmacokinetics and biodistribution in biological samples. While nanomaterials administered to the lungs may access the lymphatics via direct absorption from the airways or after initial uptake by alveolar macrophages, only 0.3 and 0.001% of the 3H-lipid dose was recovered in lung lymph fluid and lymph cell pellets (containing immune cells) respectively over 5 days. This suggests limited lymphatic access of liposomes, despite apparent pulmonary bioavailability of the 3H-lipid being approximately 17%, likely a result of absorption of liberated 3H-lipid after breakdown of the liposome in the presence of lung surfactant. Similarly, biodistribution of 3H in the mediastinal lymph node was insignificant after 5 days. These data suggest that liposomes, that are normally absorbed via the lymphatics after interstitial administration, do not access the lung lymphatics after inhaled administration. Alternate approaches to maximize the lung lymphatic delivery of drugs and other therapeutics need to be identified.

Identification of placental androgen receptor isoforms in a sheep model of maternal allergic asthma.

Maternal asthma is known to impact intrauterine growth outcomes, which may be mediated, in part, by altered androgen signalling. Our aim was to explore whether the sheep placenta expresses androgen receptor (AR) isoforms and determine if the differential expression of AR protein isoforms is altered by maternal asthma. Four known AR isoforms were detected (AR-FL, AR-v1, AR-v7, and AR-45), and their expression and subcellular distribution was altered in the presence of maternal allergic asthma. These findings underscore the importance for in vivo models of maternal asthma to delineate molecular patterns that may contribute to feto-placental growth and development.

Pulmonary Deposition of Radionucleotide-Labeled Palivizumab: Proof-of-Concept Study.

OBJECTIVE: Current prevention and/or treatment options for respiratory syncytial virus (RSV) infections are limited as no vaccine is available. Prophylaxis with palivizumab is very expensive and requires multiple intramuscular injections over the RSV season. Here we present proof-of-concept data using nebulized palivizumab delivery as a promising new approach for the prevention or treatment of severe RSV infections, documenting both aerosol characteristics and pulmonary deposition patterns in the lungs of lambs. DESIGN: Prospective animal study. SETTING: Biosecurity Control Level 2-designated large animal research facility at the Murdoch Children's Research Institute, Melbourne, Australia. SUBJECTS: Four weaned Border-Leicester/Suffolk lambs at 5 months of age. INTERVENTIONS: Four lambs were administered aerosolized palivizumab conjugated to Tc-99m, under gaseous anesthesia, using either the commercially available AeroNeb Go® or the investigational HYDRA device, placed in-line with the inspiratory limb of a breathing circuit. Lambs were scanned in a single-photon emission computed tomography (SPECT/CT) scanner in the supine position during the administration procedure. MEASUREMENTS AND MAIN RESULTS: Both the HYDRA and AeroNeb Go® produced palivizumab aerosols in the 1-5 µm range with similar median (geometric standard deviation and range) aerosol droplet diameters for the HYDRA device (1.84 ± 1.40 µm, range = 0.54-5.41µm) and the AeroNeb Go® (3.07 ± 1.56 µm, range = 0.86-10 µm). Aerosolized palivizumab was delivered to the lungs at 88.79-94.13% of the total aerosolized amount for all lambs, with a small proportion localized to either the trachea or stomach. No difference between devices were found. Pulmonary deposition ranged from 6.57 to 9.25% of the total dose of palivizumab loaded in the devices, mostly in the central right lung. CONCLUSIONS: Aerosolized palivizumab deposition patterns were similar in all lambs, suggesting a promising approach in the control of severe RSV lung infections.

Blood and saliva-derived exomes from healthy Caucasian subjects do not display overt evidence of somatic mosaicism.

Somatic mosaicism is a normal occurrence during development in the tissues and organs. As part of establishing a "healthy population "(HP) background or base-line, we investigated whether such mosaicism can be routinely detected in the circulating DNA secured from a rigorously designed healthy human liquid biopsy clinical trial (saliva, blood). We deployed next generation (NG) whole exome sequencing (WES) at median exome coverage rates of 97.2 % (-to-30x) and 70.0 % (-to-100x). We found that somatic mosaicism is not detectable by such standard bulk WES sequencing assays in saliva and blood DNA in 24 normal healthy Caucasians of both sexes from 18 to 60 years of age. We conclude that for circulating DNA using standard WES no novel somatic mutational variants can be detected in protein-coding regions of normal healthy subjects. This implies that the extent within normal tissues of somatic mosaicism must be at a lower level, below the detection threshold, for these circulating DNA WES read depths.

Aerosol Pirfenidone Pharmacokinetics after Inhaled Delivery in Sheep: a Viable Approach to Treating Idiopathic Pulmonary Fibrosis.

PURPOSE: Inhaled delivery of pirfenidone to the lungs of patients with idiopathic pulmonary fibrosis holds promise to eliminate oral-observed side effects while enhancing efficacy. This study aimed to comprehensively describe the pulmonary pharmacokinetics of inhaled aerosol pirfenidone in healthy adult sheep. METHODS: Pirfenidone concentrations were evaluated in plasma, lung-derived lymph and epithelial lining fluid (ELF) with data subjected to non-compartmental pharmacokinetic analysis. RESULTS: Compartmental pharmacokinetic evaluation indicated that a 49 mg lung-deposited dose delivered an ELF Cmax of 62 ± 23 mg/L, and plasma Cmax of 3.1 ± 1.7 mg/L. Further analysis revealed that plasma pirfenidone reached Tmax faster and at higher concentrations than in lymph. These results suggested inhaled pirfenidone was cleared from the alveolar interstitium via blood faster than the drug could equilibrate between the lung interstitial fluid and lung lymphatics. However, the data also suggested that a 'reservoir' of pirfenidone feeds into lung lymph at later time points (after it has largely been cleared from plasma), prolonging lung lymphatic exposure. CONCLUSIONS: This study indicates inhaled pirfenidone efficiently deposits in ELF and is cleared from the lungs by initial absorption into plasma, followed by later equilibrium with lung interstitial and lymph fluid.

Placental glucocorticoid receptor isoforms in a sheep model of maternal allergic asthma.

Maternal asthma increases the risk of adverse pregnancy outcomes and may affect fetal growth and placental function by differential effects on the expression of glucocorticoid receptor (GR) isoforms, leading to altered glucocorticoid signalling. Our aim was to examine the effect of maternal asthma on placental GR profiles using a pregnant sheep model of asthma. Nine known GR isoforms were detected. There was a significant increase in the expression of placental GR isoforms that are known to have low trans-activational activity in other species including GR A, GR P and GRγ which may result in a pro-inflammatory environment in the presence of allergic asthma.

Maternal allergic asthma during pregnancy alters fetal lung and immune development in sheep: potential mechanisms for programming asthma and allergy.

KEY POINTS: Experimental maternal allergic asthma in sheep provides an experimental model in which to test impacts on progeny. Fetuses from allergic asthmatic ewes had fewer surfactant-producing cells in lungs. A greater proportion of lymphocytes from thymus were CD44 positive in fetuses from allergic asthmatic ewes than in controls. These changes to fetal development might contribute to poor neonatal lung function and increased risk of allergy seen in offspring of pregnancies complicated by asthma. ABSTRACT: Asthma is prevalent in pregnancy and increases the risk of disease in offspring, including neonatal respiratory distress and childhood asthma and allergy, but the mechanisms are not understood. We hypothesized that fetal lung structure and immune phenotype in late gestation fetal sheep would be impaired in our sheep model of maternal allergic asthma during pregnancy. Singleton-bearing ewes were either sensitized before pregnancy to house dust mite (HDM, allergic, n = 7) or were non-allergic (control, n = 5). The ewes were subsequently subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Tissues were collected at 140 ± 1 days gestational age (term, ∼147 days). The density of type II alveolar epithelial cells (surfactant protein C-immunostained) in the lungs was 30% lower in fetuses from allergic ewes than in controls (P < 0.001), but tissue-to-air space ratio and numbers of leucocytes and macrophages were not different between groups. The proportion of CD44+ lymphocytes in the fetal thymus was 3.5-fold higher in fetuses from allergic ewes than in control ewes (P = 0.043). Fewer surfactant-producing type II alveolar epithelial cells may contribute to the increased risk of neonatal respiratory distress in infants of asthmatic mothers, suggesting that interventions to promote lung maturation could improve their neonatal outcomes. If the elevated lymphocyte expression of CD44 persists postnatally, this would confer greater susceptibility to allergic diseases in progeny of asthmatic mothers, consistent with observations in humans. Further experiments are needed to evaluate postnatal phenotypes of progeny and investigate potential interventions.

Umbilical cord blood versus mesenchymal stem cells for inflammation-induced preterm brain injury in fetal sheep.

BACKGROUND: Chorioamnionitis and fetal inflammation are principal causes of neuropathology detected after birth, particularly in very preterm infants. Preclinical studies show that umbilical cord blood (UCB) cells are neuroprotective, but it is uncertain if allogeneic UCB cells are a feasible early intervention for preterm infants. In contrast, mesenchymal stem cells (MSCs) are more readily accessible and show strong anti-inflammatory benefits. We aimed to compare the neuroprotective benefits of UCB versus MSCs in a large animal model of inflammation-induced preterm brain injury. We hypothesized that MSCs would afford greater neuroprotection. METHODS: Chronically instrumented fetal sheep at 0.65 gestation received intravenous lipopolysaccharide (150 ng; 055:B5, n = 8) over 3 consecutive days; or saline for controls (n = 8). Cell-treated animals received 108 UCB mononuclear cells (n = 7) or 107 umbilical cord MSCs (n = 8), intravenously, 6 h after the final lipopolysaccharide dose. Seven days later, cerebrospinal fluid and brain tissue was collected for analysis. RESULTS: Lipopolysaccharide induced neuroinflammation and apoptosis, and reduced the number of mature oligodendrocytes. MSCs reduced astrogliosis, but UCB did not have the same effect. UCB significantly decreased cerebral apoptosis and protected mature myelinating oligodendrocytes, but MSCs did not. CONCLUSION: UCB appears to better protect white matter development in the preterm brain in response to inflammation-induced brain injury in fetal sheep.

Human Umbilical Cord Blood Therapy Protects Cerebral White Matter from Systemic LPS Exposure in Preterm Fetal Sheep.

BACKGROUND: Infants born preterm following exposure to in utero inflammation/chorioamnionitis are at high risk of brain injury and life-long neurological deficits. In this study, we assessed the efficacy of early intervention umbilical cord blood (UCB) cell therapy in a large animal model of preterm brain inflammation and injury. We hypothesised that UCB treatment would be neuroprotective for the preterm brain following subclinical fetal inflammation. METHODS: Chronically instrumented fetal sheep at 0.65 gestation were administered lipopolysaccharide (LPS, 150 ng, 055:B5) intravenously over 3 consecutive days, followed by 100 million human UCB mononuclear cells 6 h after the final LPS dose. Controls were administered saline instead of LPS and cells. Ten days after the first LPS dose, the fetal brain and cerebrospinal fluid were collected for analysis of subcortical and periventricular white matter injury and inflammation. RESULTS: LPS administration increased microglial aggregate size, neutrophil recruitment, astrogliosis and cell death compared with controls. LPS also reduced total oligodendrocyte count and decreased mature myelinating oligodendrocytes. UCB cell therapy attenuated cell death and inflammation, and recovered total and mature oligodendrocytes, compared with LPS. CONCLUSIONS: UCB cell treatment following inflammation reduces preterm white matter brain injury, likely mediated via anti-inflammatory actions.

Late-gestation maternal dietary methyl donor and cofactor supplementation in sheep partially reverses protection against allergic sensitization by IUGR.

Perinatal exposures are associated with altered risks of childhood allergy. Human studies and our previous work suggest that restricted growth in utero (IUGR) is protective against allergic disease. The mechanisms are not clearly defined, but reduced fetal abundance and altered metabolism of methyl donors are hypothesized as possible underlying mechanisms. Therefore, we examined whether late-gestation maternal dietary methyl donor and cofactor supplementation of the placentally restricted (PR) sheep pregnancy would reverse allergic protection in progeny. Allergic outcomes were compared between progeny from control pregnancies (CON; n = 49), from PR pregnancies without intervention (PR; n = 28), and from PR pregnancies where the dam was fed a methyl donor plus cofactor supplement from day 120 of pregnancy until delivery (PR + Methyl; n = 25). Both PR and PR + Methyl progeny were smaller than CON; supplementation did not alter birth size. PR was protective against cutaneous hypersensitivity responses to ovalbumin (OVA; P < 0.01 in singletons). Cutaneous hypersensitivity responses to OVA in PR + Methyl progeny were intermediate to and not different from the responses of CON and PR sheep. Cutaneous hypersensitivity responses to house dust mites did not differ between treatments. In singleton progeny, upper dermal mast cell density was greater in PR + Methyl than in PR or CON (each P < 0.05). The differences in the cutaneous allergic response were not explained by treatment effects on circulating immune cells or antibodies. Our results suggest that mechanisms underlying in utero programming of allergic susceptibility by IUGR and methyl donor availability may differ and imply that late-gestation methyl donor supplementation may increase allergy risk.

Exacerbation of Ventilation-Induced Lung Injury and Inflammation in Preterm Lambs by High-Dose Nanoparticles.

Mechanical ventilation of preterm neonates causes lung inflammation and injury, with potential life-long consequences. Inert 50-nm polystyrene nanoparticles (PS50G) reduce allergic inflammation in the lungs of adult mice. We aimed to confirm the anti-inflammatory effects of PS50G in a sheep asthma model, and investigate the effects of prophylactic administration of PS50G on ventilation-induced lung injury (VILI) in preterm lambs. We assessed lung inflammatory cell infiltration, with and without PS50G, after airway allergen challenge in ewes sensitised to house dust mite. Preterm lambs (0.83 gestation) were delivered by caesarean section for immediate tissue collection (n = 5) or ventilation either with (n = 6) or without (n = 5) prophylactic intra-tracheal administration of PS50G nanoparticles (3% in 2 ml). Ventilation was continued for a total of 2 h before tissue collection for histological and biomolecular assessment of lung injury and inflammation. In ewes with experimental asthma, PS50G decreased eosinophilic infiltration of the lungs. Ventilated preterm lambs showed molecular and histological signs of lung injury and inflammation, which were exacerbated in lambs that received PSG50G. PS50G treatment decreased established inflammation in the lungs of asthmatic sheep. However, prophylactic administration of PSG50 exacerbated ventilation-induced lung injury and lung inflammation in preterm lambs.

Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs.

Ventilation of preterm neonates causes pulmonary inflammation that can contribute to lung injury, propagate systemically and result in long-term disease. Modulation of this initial response may reduce lung injury and its sequelae. We aimed to determine the effect of human amnion epithelial cells (hAECs) on immune activation and lung injury in preterm neonatal lambs. Preterm lambs received intratracheal hAECs (90x106) or vehicle, prior to 2 h of mechanical ventilation. Within 5 min of ventilation onset, lambs also received intravenous hAECs (90x106) or vehicle. Lung histology, bronchoalveolar lavage (BAL) cell phenotypes, and cytokine profiles were examined after 2 h of ventilation, and in unventilated controls. Histological indices of lung injury were higher than control, in vehicle-treated ventilated lambs but not in hAEC-treated ventilated lambs. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1ß and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease.

Substantial Targeting Advantage Achieved by Pulmonary Administration of Colistin Methanesulfonate in a Large-Animal Model.

Colistin, administered as its inactive prodrug colistin methanesulfonate (CMS), is often used in multidrug-resistant Gram-negative pulmonary infections. The CMS and colistin pharmacokinetics in plasma and epithelial lining fluid (ELF) following intravenous and pulmonary dosing have not been evaluated in a large-animal model with pulmonary architecture similar to that of humans. Six merino sheep (34 to 43 kg body weight) received an intravenous or pulmonary dose of 4 to 8 mg/kg CMS (sodium) or 2 to 3 mg/kg colistin (sulfate) in a 4-way crossover study. Pulmonary dosing was achieved via jet nebulization through an endotracheal tube cuff. CMS and colistin were quantified in plasma and bronchoalveolar lavage fluid (BALF) samples by high-performance liquid chromatography (HPLC). ELF concentrations were calculated via the urea method. CMS and colistin were comodeled in S-ADAPT. Following intravenous CMS or colistin administration, no concentrations were quantifiable in BALF samples. Elimination clearance was 1.97 liters/h (4% interindividual variability) for CMS (other than conversion to colistin) and 1.08 liters/h (25%) for colistin. On average, 18% of a CMS dose was converted to colistin. Following pulmonary delivery, colistin was not quantifiable in plasma and CMS was detected in only one sheep. Average ELF concentrations (standard deviations [SD]) of formed colistin were 400 (243), 384 (187), and 184 (190) mg/liter at 1, 4, and 24 h after pulmonary CMS administration. The population pharmacokinetic model described well CMS and colistin in plasma and ELF following intravenous and pulmonary administration. Pulmonary dosing provided high ELF and low plasma colistin concentrations, representing a substantial targeting advantage over intravenous administration. Predictions from the pharmacokinetic model indicate that sheep are an advantageous model for translational research.

A Comparison of the Pharmacokinetics and Pulmonary Lymphatic Exposure of a Generation 4 PEGylated Dendrimer Following Intravenous and Aerosol Administration to Rats and Sheep.

PURPOSE: Cancer metastasis to pulmonary lymph nodes dictates the need to deliver chemotherapeutic and diagnostic agents to the lung and associated lymph nodes. Drug conjugation to dendrimer-based delivery systems has the potential to reduce toxicity, enhance lung retention and promote lymphatic distribution in rats. The current study therefore evaluated the pharmacokinetics and lung lymphatic exposure of a PEGylated dendrimer following inhaled administration. METHODS: Plasma pharmacokinetics and disposition of a 22 kDa PEGylated dendrimer were compared after aerosol administration to rats and sheep. Lung-derived lymph could not be sampled in rats and so lymphatic transport of the dendrimer from the lung was assessed in sheep. RESULTS: Higher plasma concentrations were achieved when dendrimer was administered to the lungs of rats as a liquid instillation when compared to an aerosol. Plasma pharmacokinetics were similar between sheep and rats, although some differences in disposition patterns were evident. Unexpectedly, less than 0.5% of the aerosol dose was recovered in pulmonary lymph. CONCLUSIONS: The data suggest that rats provide a relevant model for assessing the pharmacokinetics of inhaled macromolecules prior to evaluation in larger animals, but that the pulmonary lymphatics are unlikely to play a major role in the absorption of nanocarriers from the lungs.

Development of an experimental model of maternal allergic asthma during pregnancy.

Maternal asthma during pregnancy adversely affects pregnancy outcomes but identification of the cause/s, and the ability to evaluate interventions, is limited by the lack of an appropriate animal model. We therefore aimed to characterise maternal lung and cardiovascular responses and fetal-placental growth and lung surfactant levels in a sheep model of allergic asthma. Immune and airway functions were studied in singleton-bearing ewes, either sensitised before pregnancy to house dust mite (HDM, allergic, n = 7) or non-allergic (control, n = 5), and subjected to repeated airway challenges with HDM (allergic group) or saline (control group) throughout gestation. Maternal lung, fetal and placental phenotypes were characterised at 140 ± 1 days gestational age (term, ∼147 days). The eosinophil influx into lungs was greater after HDM challenge in allergic ewes than after saline challenge in control ewes before mating and in late gestation. Airway resistance increased throughout pregnancy in allergic but not control ewes, consistent with increased airway smooth muscle in allergic ewes. Maternal allergic asthma decreased relative fetal weight (-12%) and altered placental phenotype to a more mature form. Expression of surfactant protein B mRNA was 48% lower in fetuses from allergic ewes than controls, with a similar trend for surfactant protein D. Thus, allergic asthma in pregnant sheep modifies placental phenotype, and inhibits fetal growth and lung development consistent with observations from human pregnancies. Preconceptional allergen sensitisation and repeated airway challenges in pregnant sheep therefore provides an animal model to identify mechanisms of altered fetal development and adverse pregnancy outcomes caused by maternal asthma in pregnancy.

Dynamics of IL-4 and IL-13 expression in the airways of sheep following allergen challenge.

BACKGROUND: IL-4 and IL-13 play a critical yet poorly understood role in orchestrating the recruitment and activation of effector cells of the asthmatic response and driving the pathophysiology of allergic asthma. The house dust mite (HDM) sheep asthma model displays many features of the human condition and is an ideal model to further elucidate the involvement of these critical Th2 cytokines. We hypothesized that airway exposure to HDM allergen would induce or elevate the expression profile of IL-4 and IL-13 during the allergic airway response in this large animal model of asthma. METHODS: Bronchoalveolar lavage (BAL) samples were collected from saline- and house dust mite (HDM)- challenged lung lobes of sensitized sheep from 0 to 48 h post-challenge. BAL cytokines (IL-4, IL-13, IL-6, IL-10, TNF-α) were each measured by ELISA. IL-4 and IL-13 expression was assessed in BAL leukocytes by flow cytometry and in airway tissue sections by immunohistology. RESULTS: IL-4 and IL-13 were increased in BAL samples following airway allergen challenge. HDM challenge resulted in a significant increase in BAL IL-4 levels at 4 h compared to saline-challenged airways, while BAL IL-13 levels were elevated at all time-points after allergen challenge. IL-6 levels were maintained following HDM challenge but declined after saline challenge, while HDM administration resulted in an acute elevation in IL-10 at 4 h but no change in TNF-α levels over time. Lymphocytes were the main early source of IL-4, with IL-4 release by alveolar macrophages (AMs) prominent from 24 h post-allergen challenge. IL-13 producing AMs were increased at 4 and 24 h following HDM compared to saline challenge, and tissue staining provided evidence of IL-13 expression in airway epithelium as well as immune cells in airway tissue. CONCLUSION: In a sheep model of allergic asthma, airway inflammation is accompanied by the temporal release of key cytokines following allergen exposure that primarily reflects the Th2-driven nature of the immune response in asthma. The present study demonstrates for the first time the involvement of IL-4 and IL-13 in a relevant large animal model of allergic airways disease.

The innate response to peanut extract in ovine afferent lymph and its correlation with allergen sensitisation.

The innate response generated after initial allergen exposure is crucial for polarising adaptive immunity, but little is known about how it drives an atopic or type-2 immune response. The present study characterises the response of skin-draining afferent lymph in sheep following injection with peanut (PN) extract in the presence or absence of aluminium hydroxide (AlOH) adjuvant. Lymph was collected and innate cell populations characterised over an 84 h time period. The innate response to PN extract in afferent lymph displayed an early increase in neutrophils and monocytes without any changes in the dendritic cell (DC) population. PN antigen was transported by neutrophils and monocytes for the first 36 h, after which time DCs were the major antigen trafficking cells. AlOH adjuvant gradually increased antigen uptake by DCs at the later time points. Following lymphatic characterisation, sheep were sensitised with PN extract by three subcutaneous injections of PN in AlOH, and the level of PN-specific immunoglobulin E (IgE) was determined. Sheep with higher levels of steady-state DCs in afferent lymph showed increased monocytic recruitment in afferent lymph and reduced PN-specific IgE following sensitisation. In addition, DCs from afferent lymph that had ingested PN antigen increased the expression of monocyte chemoattractant mRNA. The results of this study show that the innate response to PN extract involves a dynamic change in cell populations in the afferent lymph over time. In addition, DCs may determine the strength of the initial inflammatory cell response, which in turn may determine the nature of the antigen-specific adaptive response.

Placental restriction of fetal growth reduces cutaneous responses to antigen after sensitization in sheep.

Prenatal and early childhood exposures are implicated as causes of allergy, but the effects of intrauterine growth restriction on immune function and allergy are poorly defined. We therefore evaluated effects of experimental restriction of fetal growth on immune function and allergic sensitization in adolescent sheep. Immune function (circulating total red and white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils, and the antibody response to Clostridial vaccination) and responses to house dust mite (HDM) allergen and ovalbumin (OVA) antigen sensitization (specific total Ig, IgG1, and IgE antibodies, and cutaneous hypersensitivity) were investigated in adolescent sheep from placentally restricted (PR, n = 23) and control (n = 40) pregnancies. Increases in circulating HDM-specific IgE (P = 0.007) and OVA-specific IgE (P = 0.038) were greater in PR than control progeny. PR did not alter total Ig, IgG1, or IgM responses to either antigen. PR increased OVA-specific but not HDM-specific IgA responses in females only (P = 0.023). Multiple birth increased Ig responses to OVA in a sex-specific manner. PR decreased the proportion of positive cutaneous hypersensitivity responders to OVA at 24 h (P = 0.030) but had no effect on cutaneous responses to HDM. Acute wheal responses to intradermal histamine correlated positively with birth weight in singletons (P = 0.023). Intrauterine growth restriction may suppress inflammatory responses in skin downstream of IgE induction, without impairment in antibody responses to a nonpolysaccharide vaccine. Discord between cutaneous and IgE responses following sensitization suggests new mechanisms for prenatal allergy programming.