Wayne Mitzner

Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease

Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants. A formalin-inactivated RSV vaccine was used to immunize children in 1966 and elicited non-protective, pathogenic antibody. Two immunized infants died and 80% were hospitalized after subsequent RSV exposure. No vaccine was licensed since. A widely accepted hypothesis attributed vaccine failure to formalin disruption of protective antigens. Instead, we show that lack of protection was not due to alterations caused by formalin, but to low antibody avidity for protective epitopes. Lack of antibody affinity maturation followed poor Toll-like receptor stimulation. This study explains why the inactivated RSV vaccine failed to protect and consequently led to severe disease, hampering vaccine development for forty-two years. Also, it suggests that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR-agonists in their formulation. In addition, it identifies affinity maturation as a critical factor for the safe immunization of infants.Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants. A formalin-inactivated RSV vaccine was used to immunize children and elicited nonprotective, pathogenic antibody. Immunized infants experienced increased morbidity after subsequent RSV exposure. No vaccine has been licensed since that time. A widely accepted hypothesis attributed the vaccine failure to formalin disruption of protective antigens. Here we show that the lack of protection was not due to alterations caused by formalin but instead to low antibody avidity for protective epitopes. Lack of antibody affinity maturation followed poor Toll-like receptor (TLR) stimulation. This study explains why the inactivated RSV vaccine did not protect the children and consequently led to severe disease, hampering vaccine development for 42 years. It also suggests that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR agonists in their formulation, and it identifies affinity maturation as a key factor for the safe immunization of infants.

Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma. As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling. Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM. In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice.

An increased airway response to various bronchocon‐stricting agents is one of the hallmarks of asthma. An interdependence of heredity and environment appears to determine this nonspecific hyperreactivity of the airways. The present study describes the patterns of inheritance of the airway response to a direct mediator of smooth muscle contraction (acetylcholine) in A/J and C3H/HeJ inbred mice and their offspring. The mean airway response to acetylcholine was greater than sixfold higher in A/J mice as compared with C3H/HeJ mice. Two phenotypes were easily distinguished on the basis of airway responses to acetylcholine in the progeny of A/J and C3H/HeJ mice. These two phenotypes were termed HYPERREACTIVE (after the A/J strain) and HYPOREACTIVE (after the C3H/HeJ strain). The observed frequencies of HYPERREACTIVE and HYPOREACTIVE phenotypes in the (A/J × C3H/HeJ) Fl; (C3H/HeJ × A/J) F1 × C3H/HeJ (C3H/HeJ backcross); and the [(A/J × C3H/HeJ) F1 × (C3H/HeJ x A/J) F1] F2 are consistent with a single autosomal recessive gene primarily controlling acetylcholine‐mediated airway responses. This single gene difference in airway response is completely inhibited by atropine and therefore mediated entirely by the muscarinic acetylcholine receptor.— Levitt, R. C.; Mitzner, W. Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice. FASEB J. 2: 2605‐2608; 1988.

Telomere Length Is a Determinant of Emphysema Susceptibility

RATIONALE Germline mutations in the enzyme telomerase cause telomere shortening, and have their most common clinical manifestation in age-related lung disease that manifests as idiopathic pulmonary fibrosis. Short telomeres are also a unique heritable trait that is acquired with age. OBJECTIVES We sought to understand the mechanisms by which telomerase deficiency contributes to lung disease. METHODS We studied telomerase null mice with short telomeres. MEASUREMENTS AND MAIN RESULTS Although they have no baseline histologic defects, when mice with short telomeres are exposed to chronic cigarette smoke, in contrast with controls, they develop emphysematous air space enlargement. The emphysema susceptibility did not depend on circulating cell genotype, because mice with short telomeres developed emphysema even when transplanted with wild-type bone marrow. In lung epithelium, cigarette smoke exposure caused additive DNA damage to telomere dysfunction, which limited their proliferative recovery, and coincided with a failure to down-regulate p21, a mediator of cellular senescence, and we show here, a determinant of alveolar epithelial cell cycle progression. We also report early onset of emphysema, in addition to pulmonary fibrosis, in a family with a germline deletion in the Box H domain of the RNA component of telomerase. CONCLUSIONS Our data indicate that short telomeres lower the threshold of cigarette smoke-induced damage, and implicate telomere length as a genetic susceptibility factor in emphysema, potentially contributing to its age-related onset in humans.

Radiofrequency ablation of airway smooth muscle for sustained treatment of asthma: preliminary investigations

Bronchial thermoplasty is a procedure now being tested in humans for the treatment of asthma. Current studies focusing on safety are encouraging. The procedure, which causes extensive ablation of airway smooth muscle (ASM), is well tolerated, and there is a sustained reduction in airway responsiveness to methacholine. Two assumptions underlie the development of this procedure: 1) ASM is a vestigial tissue; and 2) that treatment directed at ASM alone will provide sustained symptomatic and physiological improvement in asthmatic humans. Even if this procedure is efficacious, it must be safe in the long-term. Current studies in animals and humans suggest that this is very likely to be the case. While bronchial thermoplasty may have a broad application, especially for patients who wish for a permanent amelioration of their symptoms or have difficulty adhering to medical regimens, the compelling use of this procedure is for patients who are inadequately controlled on current drug therapy or who cannot adhere to therapeutic regimens. The application of this procedure for the treatment of asthma is currently being considered by regulatory agencies, and study centres are currently disseminated throughout North America and Europe. Within the next 1–2 yrs, a profile of the potential role of this therapy in human asthma should be developed fully.

Angiogenesis in the Mouse Lung

When pulmonary arterial blood flow is obstructed in all mammals studied, there is a compensatory growth of the bronchial vasculature. This angiogenesis normally occurs through a proliferation of the systemic circulation to the intraparenchymal airways. It is an important pathophysiological process, not only in pulmonary vascular disease, but also in lung cancer, because the blood flow that supplies primary lung tumors arises from the systemic circulation. In the mouse, however, the systemic blood vessels that supply the trachea and mainstem bronchi do not penetrate into the intraparenchymal airways, as they do in all other larger species. In this study, we attempted to generate a new functional bronchial circulation in the mouse by permanently obstructing 40% of the pulmonary circulation. We quantified the systemic blood flow to the lung with fluorescent microspheres for 3 months after left pulmonary artery ligation. Results demonstrated that a substantial systemic blood flow to the lung that can eventually supply up to 15% of the normal pulmonary flow can be generated beginning 5-6 days after ligation. These new angiogenic vessels do not arise from the extraparenchymal bronchial circulation. Rather they enter the lung directly via a totally new vasculature that develops between the visceral and parietal pleuras, supplied by several intercostal arteries. This unique model of angiogenesis occurs in the absence of any hypoxic stimulus and mimics the vascular source of many lung tumors.

Invasive and noninvasive methods for studying pulmonary function in mice

The widespread use of genetically altered mouse models of experimental asthma has stimulated the development of lung function techniques in vivo to characterize the functional results of genetic manipulations. Here, we describe various classical and recent methods of measuring airway responsiveness in vivo including both invasive methodologies in anesthetized, intubated mice (repetitive/non-repetitive assessment of pulmonary resistance (RL) and dynamic compliance (Cdyn); measurement of low-frequency forced oscillations (LFOT)) and noninvasive technologies in conscious animals (head-out body plethysmography; barometric whole-body plethysmography). Outlined are the technical principles, validation and applications as well as the strengths and weaknesses of each methodology. Reviewed is the current set of invasive and noninvasive methods of measuring murine pulmonary function, with particular emphasis on practical considerations that should be considered when applying them for phenotyping in the laboratory mouse.

Telomere dysfunction causes alveolar stem cell failure

Significance Idiopathic pulmonary fibrosis and emphysema are leading causes of mortality, but there are no effective therapies. Mutations in telomerase are the most common identifiable risk factor for idiopathic pulmonary fibrosis. They also predispose to severe emphysema in smokers, occurring at a frequency similar to α-1 antitrypsin deficiency. The work shown here points to alveolar stem cell senescence as a driver of these pathologies. Epithelial stem cell failure was associated with secondary inflammatory recruitment and exquisite susceptibility to injury from “second hits.” The findings suggest that efforts to reverse the stem cell failure state directly, rather than its secondary consequences, may be an effective therapy approach in telomere-mediated lung disease. Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.

Betamethasone and the rhesus fetus: effect on lung morphometry and connective tissue.

Summary: Pregnant rhesus monkeys (Macaca mulatta) at 67 to 85% of term pregnancy were treated with betamethasone for 3 days and then delivered by cesarean section. These treated fetuses had larger lung volumes (32.6 × 1.8 ml/kg of body weight) compared to gestational age-matched controls (22.9 × 3.2 ml/kg of body weight; P < 0.025) but no alterations in surfactant properties as measured by amniotic fluid L/S ratios, alveolar deflation stability, or lung phosphatidylcholine. These findings suggest that betamethasone effects an increase in fetal lung volume by some method other than alteration in alveolar surfactant concentrations. Results also demonstrated an 11% increase in the collagen to elastin concentration in the treated fetuses as compared to the control animals (P < 0.01), suggesting alterations in lung connective tissue. Morphometric studies done on the air-fixed inflated lung demonstrated a decrease in the number of alveoli per unit volume of lung among the treated animals (0.95 × 0.07 × 106) compared to the control animals (1.19 × 0.08 × 106; P < 0.025) and a reduction in the mean surface area of the lungs of the treated animals (506 × 10 cm2 per cm3) compared to the control animals (561 × 9 cm2 per cm3; P < 0.005). These findings suggest that at least part of the increased maximal lung volumes is related to increased alveolar distensibility. Together, these pressure volume findings, biochemical studies, and morphometric analyses indicate that a major effect of betamethasone on the rhesus fetal lung is to alter lung connective tissue characteristics. Alterations in lung surfactant appear to be of less functional significance in this rhesus fetal model. The disparity between these findings and other animal studies might be due to differences in species, the preparation, or the method of glucocorticoid administration.Speculation: Maternal betamethasone treatment produces alterations in the connective tissue properties of the developing fetal lung, which could have long-lasting effects on lung mechanical properties.

Angiotensin receptor blockade attenuates cigarette smoke–induced lung injury and rescues lung architecture in mice

Chronic obstructive pulmonary disease (COPD) is a prevalent smoking-related disease for which no disease-altering therapies currently exist. As dysregulated TGF-β signaling associates with lung pathology in patients with COPD and in animal models of lung injury induced by chronic exposure to cigarette smoke (CS), we postulated that inhibiting TGF-β signaling would protect against CS-induced lung injury. We first confirmed that TGF-β signaling was induced in the lungs of mice chronically exposed to CS as well as in COPD patient samples. Importantly, key pathological features of smoking-associated lung disease in patients, e.g., alveolar injury with overt emphysema and airway epithelial hyperplasia with fibrosis, accompanied CS-induced alveolar cell apoptosis caused by enhanced TGF-β signaling in CS-exposed mice. Systemic administration of a TGF-β-specific neutralizing antibody normalized TGF-β signaling and alveolar cell death, conferring improved lung architecture and lung mechanics in CS-exposed mice. Use of losartan, an angiotensin receptor type 1 blocker used widely in the clinic and known to antagonize TGF-β signaling, also improved oxidative stress, inflammation, metalloprotease activation and elastin remodeling. These data support our hypothesis that inhibition of TGF-β signaling through angiotensin receptor blockade can attenuate CS-induced lung injury in an established murine model. More importantly, our findings provide a preclinical platform for the development of other TGF-β-targeted therapies for patients with COPD.