Lung effects of inhaled corticosteroids in a rhesus monkey model of childhood asthma.

BACKGROUND: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy. OBJECTIVE: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates. METHODS: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline. RESULTS: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.

Airway generation-specific differences in the spatial distribution of immune cells and cytokines in allergen-challenged rhesus monkeys.

BACKGROUND: Accumulation of immune cell populations and their cytokine products within tracheobronchial airways contributes to the pathogenesis of allergic asthma. It has been postulated that peripheral regions of the lung play a more significant role than proximal airways with regard to inflammatory events and airflow obstruction. OBJECTIVE: To determine whether immune cell populations and associated cytokines are uniformly distributed throughout the conducting airway tree in a non-human primate model of allergic asthma. METHODS: We used a stereologic approach with a stratified sampling scheme to measure the volume density of immune cells within the epithelium and interstitium of trachea and 4-5 intrapulmonary airway generations from house dust mite (HDM) (Dermatophagoides farinae)-challenged adult monkeys. In conjunction with immune cell distribution profiles, mRNA levels for 21 cytokines/chemokines and three chemokine receptors were evaluated at four different airway generations from microdissected lungs. RESULTS: In HDM-challenged monkeys, the volume of CD1a+ dendritic cells, CD4+ T helper lymphocytes, CD25+ cells, IgE+ cells, eosinophils, and proliferating cells were significantly increased within airways. All five immune cell types accumulated within airways in unique patterns of distribution, suggesting compartmentalized responses with regard to trafficking. Although cytokine mRNA levels were elevated throughout the conducting airway tree of HDM-challenged animals, the distal airways (terminal and respiratory bronchioles) exhibited the most pronounced up-regulation. CONCLUSION: These findings demonstrate that key effector immune cell populations and cytokines associated with asthma differentially accumulate within distinct regions and compartments of tracheobronchial airways from allergen-challenged primates.

Total number and mean size of alveoli in mammalian lung estimated using fractionator sampling and unbiased estimates of the Euler characteristic of alveolar openings.

Estimation of alveolar number in the lung has traditionally been done by assuming a geometric shape and counting alveolar profiles in single, independent sections. In this study, we used the unbiased disector principle to estimate the Euler characteristic (and thereby the number) of alveolar openings in rat lungs and rhesus monkey lung lobes and to obtain robust estimates of average alveolar volume. The estimator of total alveolar number was based on systematic, uniformly random sampling using the fractionator sampling design. The number of alveoli in the rat lung ranged from 17.3 x 10(6) to 24.6 x 10(6), with a mean of 20.1 x 10(6). The average number of alveoli in the two left lung lobes in the monkey ranged from 48.8 x 10(6) to 67.1 x 10(6) with a mean of 57.7 x 10(6). The coefficient of error due to stereological sampling was of the order of 0.06 in both rats and monkeys and the biological variation (coefficient of variance between individuals) was 0.15 in rat and 0.13 in monkey (left lobe, only). Between subdivisions (left/right in rat and cranial/caudal in monkey) there was an increase in variation, most markedly in the rat. With age (2-13 years) the alveolar volume increased 3-fold (as did parenchymal volume) in monkeys, but the alveolar number was unchanged. This study illustrates that use of the Euler characteristic and fractionator sampling is a robust and efficient, unbiased principle for the estimation of total alveolar number in the lung or in well-defined parts of it.

Immune and airway effects of house dust mite aeroallergen exposures during postnatal development of the infant rhesus monkey.

BACKGROUND: The effect of chronic environmental aeroallergen exposure on the immune system and airways has not been experimentally defined in very young children. OBJECTIVE: The purpose of this study was to determine the immunophenotype of peripheral blood and airway leucocytes in the newborn rhesus macaque monkey, following recurrent aerosol exposure to house dust mite (HDM) (Dermatophagoides farinae). METHODS: A regimen of HDM aerosolization was initiated for 2 h per day, three times per week, starting when rhesus macaque monkeys were 1 week of age. All monkeys were inoculated with diptheria, tetanus, and acellular pertussis vaccine at 5 weeks of age to simulate human infant vaccination schedules. RESULTS: Following 8 weeks of HDM aeroallergen exposure, infant monkeys exhibited a significant reduction in the total peripheral blood lymphocyte numbers and a decreased frequency of peripheral blood CD4+ T lymphocytes with a CD45RA-'memory' immunophenotype. Lavage CD4+ T lymphocytes from HDM-exposed monkeys showed elevated expression of CD25, as well as an increase in CD45RA-/CD62L-/CD11ahigh immunophenotype. Eosinophils were more abundant within airways of HDM-exposed monkeys, accumulating maximally within the trachea. CONCLUSION: These data demonstrate the development of immunological responses following chronic inhalation of a common environmental allergen during postnatal maturation in the non-human primate.

Heterogeneity of clara cell glutathione. A possible basis for differences in cellular responses to pulmonary cytotoxicants.

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.

Pasteurella multocida enters polarized epithelial cells by interacting with host F-actin.

We investigated the interaction of an avian strain of Pasteurella multocida with the cytoskeleton of MDCK cells, which formed a polarized epithelium when grown on type I collagen coated filters. Bacteria were incubated with MDCK cells for 30 min. 2, 4 and 6 hours and their location and association with the cell cytoskeleton determined by double-label immunofluorescence confocal microscopy. Cells were stained with a polyclonal antiserum to the outer-membrane proteins of P. multocida and with rhodamine phalloidin which specifically binds filamentous (F) actin. Confocal microscopy revealed that bacteria entered the cells by 30 min, and that by 6 hours there was a marked alteration in the actin cytoskeleton in which long filaments were reorganized to discrete foci of short actin filaments, within which were one or more bacteria. Electron microscopy demonstrated that by 2 hours, each bacterium was associated with many short 5-6 nm filaments. Treatment of MDCK cells with cytochalasin D for either 30 minutes or 24 hours prior to infection disrupted the actin cytoskeleton and inhibited entry of P. multocida.

Antibody reactions with retina and cancer-associated antigens in 10 patients with cancer-associated retinopathy.

OBJECTIVE: Our objectives were to determine what clinical characteristics are common to the form of cancer-associated retinopathy (CAR) encountered in patients with small-cell carcinoma of the lung (SCCL). Is the 23-kd retinal CAR antigen/antibody reaction present in other forms of retinopathy? Can an antigen identical or similar to the 23-kd retinal CAR antigen be identified in an established culture of SCCL? METHODS: Ten patients with CAR who had SCCL were identified by their antibody reactivity with the 23-kd retinal CAR antigen. We inquired into common clinical characteristics by means of questionnaires to the referring physicians. We looked for antigen/antibody reactions with the 23-kd retinal CAR antigen in patients with diabetic and age-related macular degenerations and in a continuous, in vitro propagated culture of SCCL (HTB 119) obtained from the American Type Culture Collection. RESULTS: We encountered many similar signs and symptoms in our patient population. These included rapid vision loss, night blindness, color loss, vitreous cells, and either flat or greatly reduced electroretinograms. No corollary to the 23-kd CAR antigen/antibody could be identified in unrelated retinopathies or cultured SCCL. CONCLUSIONS: We conclude that patients with SCCL-related CAR consistently produce antibodies against the 23-kd retinal CAR antigen. This immunologic reaction was not found in patients with unrelated retinopathies and may possibly represent a cancer marker for SCCL.

Intraperitoneal cultivation of small-cell carcinoma induces expression of the retinal cancer-associated retinopathy antigen.

OBJECTIVE: We have inquired into the reason why patients with cancer-associated retinopathy (CAR) produce antibody reactions with the 23-kd retinal CAR antigen. Possible reasons include the expression of this antigen in the related carcinoma. Previous studies have failed to identify any antigenic counterpart expressed by in vitro cultivated small-cell carcinoma of the lung. We, therefore, inquired into the effects of in vivo cultivation of the cancer cells and its influence on protein expression, with specific reference to the appearance of the 23-kd retinal CAR antigen. DESIGN: A complementary DNA library was prepared from small-cell carcinoma of the lung cells propagated intraperitoneally in Lewis rats and probed with antibodies reactive with the 23-kd retinal CAR antigen. RESULTS: We found evidence of the expression of a cancer-associated gene in ascites-propagated small-cell carcinoma of the lung that encodes for a protein antigenically similar to the 23-kd retinal CAR antigen. A complementary DNA encoding this protein revealed complete DNA sequence homology with the retinal CAR antigen showing the cancer cells are expressing this photoreceptor protein. CONCLUSIONS: We hypothesize that the carcinoma-retina immunologic cross-reaction is responsible for the induction of the unique antibody response encountered in patients with CAR with vision loss developing as a cancer-evoked autoimmune retinopathy.

The cancer-associated retinopathy antigen is a recoverin-like protein.

Cancer-associated retinopathy (CAR) is a rare form of retinal degeneration that occurs in association with certain forms of cancer. CAR patients typically possess high titers of autoantibodies against a specific photoreceptor protein--the 23 kD retinal CAR antigen. The mechanisms involved in the vision loss experienced by CAR patients are not understood, but serologic studies indicate the process could include a series of autoimmune reactions directed at specific components of the retina. Because the retinal CAR antigen is the principal ocular autoantigen involved in the antibody response of CAR patients, characterizing it would contribute to the understanding of putative autoimmune involvement. Serum antibodies from CAR patients have been used to isolate the gene encoding the CAR antigen from a cDNA library of human retina. Nucleotide sequence analysis suggests that the CAR antigen shows approximately 90% homology to the published amino acid sequence of bovine recoverin.

Computer-assisted morphometry: point, intersection, and profile counting and three-dimensional reconstruction.

The use of computers in morphometry can involve 1) automated image analysis, semiautomated image analysis and point, intersection, intercept and profile counts of two-dimensional images on tissue sections with mathematical extrapolation to the third dimension, 2) direct measurement of volumes, surfaces, lengths, and curvature using x,y,z coordinates of serial sectioned images, or 3) stereologic techniques and serial sections which is a combination of 1 and 2 above. Automated and semiautomated image analysis are generally restricted to specimens that are characterized by differential contrast such as interalveolar septa in the lung or histochemically stained mucous granules in pulmonary epithelium. Point, intersection, and profile counts using hand-held, notebook PCs, portable PCs, or standard PCs and MS-DOS-based application programs are extremely efficient, precise, affordable, and convenient methods of quantitating average values of a population. When morphometric measurements of individual structures are required, computer-assisted three-dimensional reconstruction using x,y,z coordinates of the surface outline from serial sections is a tedious yet precise method. We describe a computer program that efficiently estimates mean caliper diameter, volume, and surface area with less than five percent error with five sections per structure. We also describe a program that does digital image subtraction on serial sections, superimposes digitally generated test systems on biological images, and accumulates point, intersection, and profile counts using a Macintosh II series computer.

Circulating and localized immune complexes in experimental mycoplasma-induced arthritis-associated ocular inflammation.

Ocular deposits of immune complexes are believed to contribute to the anterior segment inflammations observed in association with the human arthritides. Arthritis-related ocular inflammations may be reproduced in animals by infection with certain species of mycoplasma. To evaluate the role of immune complexes in the production of ocular lesions, we studied their involvement in the rodent model of experimental arthritis-associated ocular inflammation induced by Mycoplasma arthritidis. Sprague-Dawley rats were infected with viable concentrates of M. arthritidis and monitored for the production of related circulating and intraocular immune complexes. Circulating immune complexes were monitored by antigen capture systems, and localized intraocular complexes were identified by indirect immunohistochemistry. Polyacrylamide gel immunoblot analysis of captured complexes confirmed the antigen(s) involved as proteins derived from M. arthritidis. Indirect immunofluorescence revealed localized complexes containing mycoplasma antigens within the ciliary-iris vasculature. Concentrations of the generated complexes diminished rapidly over a 30-day period. While complex deposits within ocular tissues could represent a contributing cause to the localized anterior segment inflammation reported in this rodent model, secondary challenge with viable M. arthritidis, which reproduced high concentrations of intraocular and circulating immune complexes, failed to elicit any ocular response.

Management and monitoring of cancer-associated retinopathy.

Cancer-associated retinopathy is a rare paraneoplastic event that can involve allergic reactions and result in retinal degradation. A patient, who had a 35-year smoking history, complained of visual loss and was found to have serum antibodies that reacted with an extract of retina, including the previously described retinal cancer-associated retinopathy antigen. Prednisone treatment appeared to reduce the patient's antibody titers to normal levels. Visual fields stabilized, and the patient was able to maintain useful vision throughout the course of treatment until his death 1 year following initial diagnosis. To our knowledge, this is the first reported case in which monitoring of antibody responses to retinal antigens appeared to be useful in the decision whether to initiate prednisone therapy. Rising antibody titers to the cancer-associated retinopathy antigen probably occurs before progressive visual field loss and may be considered an indication for prompt steroid therapy.

Comparison of lectin reactivity in the vessel beds of the rat eye.

The capillary beds of the eye are lined by two types of endothelia, fenestrated in the choriocapillaris and ciliary body, and continuous in the retina and iris. In this study, we wished to find a marker for each of these types of vessel beds using lectin histochemistry. Sections of glutaraldehyde fixed rat eyes embedded in epoxy resin were extracted with sodium ethoxide and rehydrated. Binding of 15 different lectins was visualized using the avidin-biotin peroxidase technique. We found WGA, WGA-s, LFA and PHA-E to strongly bind retinal vessels. In addition to the above lectins, iris vessels bound GSL-I. Choriocapillaris reacted variably only with WGA and not at all with other lectins tested. Vessels of ciliary body processes did not react with any lectin studied. The less fenestrated vessels of the base of the ciliary process bound lectins similar to the retina. We speculate that the differential lectin staining of the various vessel beds of the eye may reflect the degree of fenestration of the endothelium. This reactivity may be influenced by variations in the surrounding milieu including cells and extracellular matrix.

Antibody indications of secondary and superimposed retinal hypersensitivity in retinitis pigmentosa.

Antibody reactions with recognized retinopathy-inducing retinal antigens may be interpreted to reflect ongoing autoimmune events responsible for some forms of vision loss. We sought evidence of secondary and superimposed retinal hypersensitivity indicated by such antibody reactivity in a random group of patients with retinitis pigmentosa. We identified patterns of immunologic reactivity within members of a group of 52 patients with retinitis pigmentosa, which suggests some patients with retinitis pigmentosa may experience consequential superimposed retinal hypersensitivity. Identifying subgroups of patients with retinitis pigmentosa who exhibit indications of retinal hypersensitivity to known uveitopathogenic retinal proteins may permit the reduction of their rate of retinal degradation by immunomodulation.

Alterations in glial cell morphology and glial fibrillary acidic protein expression in urethane-induced retinopathy.

Urethane injection in newborn rats causes a photoreceptor degeneration without initial damage to the retinal pigment epithelium, choriocapillaris, or inner retina. In later stages, retinal vessels become incorporated into the retinal pigment epithelium (RPE) and change from a continuous endothelial cell phenotype to a fenestrated phenotype. At the light-microscopic level, there do not appear to be morphologic changes in the inner retina up to 24 weeks of age. Ultrastructurally, however, there are alterations in Müller cell cytotopographic organization. In the normal retina, intermediate filaments are primarily found from the ganglion cell layer to the inner nuclear layer. These filaments do not show glial fibrillary acidic protein immunoreactivity (GFAP-IR) in the normal animal. In the urethane-treated animals, the compartmental organization of the Müller cell organelles is moved vitread. Intermediate filaments are found in the end-foot region, and in the inner plexiform layer, bundles of intermediate filaments become more prominent. All of these filaments are GFAP-IR positive. The new expression of GFAP in the Müller cell may be linked to the observed rearrangement of the cytoskeletal elements. In urethane-induced retinopathy, GFAP-IR is found associated with vessels in all layers of the remaining retina. However, it is not seen accompanying vessels into the RPE. Ultrastructurally, there is no glial investment of the RPE-associated vessels. This absence of glial investment may permit the change in phenotype observed in these vessels.

Selective neovascularization of the retinal pigment epithelium in rat photoreceptor degeneration in vivo.

Photoreceptor cell degeneration in rodents from a variety of causes results in neovascularization of the retinal pigment epithelium as a late stage phenomenon. Even though the vessels within the pigment epithelium arise from the retinal circulation, they can manifest the choroidal endothelial cell phenotype of fenestrated endothelial cells. In order to study the detailed cellular events which result in incorporation of retinal vessels within the retinal pigment epithelium, a morphological and morphometric analysis of the RPE and vasculature was performed in rats. Urethane, given subcutaneously to newborn rats, results in a photoreceptor degeneration but does not affect the RPE, choroid or inner retinal layers. Retinas were studied from rats of 8 to 24 weeks of age, the time period when vascularization of the RPE occurs. Loss of retinal vessels is first seen at 12 weeks, primarily in substantial dropout of vessel profiles in the outer plexiform layer (OPL) vessel bed. There is a gradient of loss from the OPL bed to the nerve fiber layer (NFL) bed and from the central to peripheral region. Total vessel density of the experimental retinas is greater than controls at 8 and 12 weeks. This occurs because there is marked loss of retinal thickness, due to photoreceptor degeneration, without a comparable loss of vessel profiles. The total retinal vessel density decreases from 8 to 20 weeks, and appears to stabilize at 20 and 24 weeks. Analysis of the separate vessel beds shows that this apparent stabilization is due to continued loss of vessels within the sensory retina, and increased presence of vascular profiles within the RPE. Total absence of the photoreceptor cell is necessary for incorporation of vessels within the RPE. Since new vessel profiles develop in the RPE but not the adjacent sensory retina, we speculate that the RPE may stimulate neovascularization of the RPE. A model of the cellular events leading to RPE neovascularization is proposed.

Interglial cell gap junctions increase in urethane-induced photoreceptor degeneration in rats.

Gap junctions are found between astrocytes in the inner retina of normal rats, but they are rare between Müller cells or between astrocytes and Müller cells in the inner retina. After photoreceptor degeneration induced by urethane treatment of newborn animals, morphologic alterations of glial cells occur in the inner retina. The Müller cells withdraw from the inner limiting membrane, and the astrocytes hypertrophy and occupy the vitread surface of the inner limiting membrane. The frequency and size of the gap junctions between astrocytes increases with time in rats with urethane-induced photoreceptor degeneration, to a greater extent than expected from elaboration of additional astrocyte plasma membrane. The gap junction-profile length per glial cell membrane-contact length is 2.8 +/- 1.1 microns/1000 microns of membrane in 8-week-old normal animals; it increases to 18.9 +/- 9.4 microns/1000 microns of membrane at 56 weeks of age in urethane-treated animals. The average size of the gap junction-profile length doubles during this same period. To the authors' knowledge this is the first study demonstrating pathologic changes in gap junctions in central nervous system tissue. The authors speculate that this up-regulation of gap junctions occurs in response to an altered extracellular ionic composition in an attempt to increase the lateral spatial buffering of K+ by these cells. The relative location of glial cells in retina can determine, in part, the vulnerability of the retina to edema.

Interactions of cultured rat synovial and ocular ciliary body cells with two strains of Mycoplasma arthritidis.

Strains of Mycoplasma arthritidis differ in their ability to cause joint and ocular inflammations. Although the reasons for this difference are not fully understood, pathogenic mycoplasmas commonly require close associations with the cells they damage. Using 3H-uridine labeled mycoplasma, we compared cellular interactions of in vitro cultivated rat synovial and ocular ciliary body epithelial cells with two American Type Culture Collection strains of M. arthriditis shown to differ in their virulence. Radiolabeling assays gave evidence of a stronger retention capability on cultured cells by the more pathogenic strain, 14152. Scanning electron microscopy demonstrated cellular associations with the two strains of mycoplasma, with more of the 14152 adhering to both cell types. Examination by transmission electron microscopy showed evidence of contact between the more virulent 14152 strain and both cell types, but no similar evidence with the comparatively less virulent strain, 19611. The pathogenicity of different strains of M. arthritidis may vary according to their ability to closely associate with specific target cells involved in the disease process.

Cytodifferentiation of two epithelial populations of the respiratory bronchiole during fetal lung development in the rhesus monkey.

This study describes the cytodifferentiation of the two populations of epithelial cells found in the respiratory bronchiole of the adult rhesus monkey. One population, pseudostratified and containing ciliated, nonciliated secretory, and basal cells, is found overlying the pulmonary artery (PA). The other population, not associated with the PA, contains nonciliated cuboidal cells between alveolar outpockets. In this study we used terminal conducting airways from the lungs of fetal (90 to 155 days gestational age [DGA]), postnatal, and adult rhesus monkeys. Ciliated cells were partially differentiated at 90 DGA (54% gestation) and completely differentiated by 134 DGA (80% gestation). Nonciliated secretory cells were partially differentiated at 95 DGA (57% gestation) but did not lose all glycogen until the postnatal period. Basal cells appeared by 134 DGA (80% gestation) and matured in the postnatal period. Small mucous granule cells appeared at 125 DGA (74% gestation) and did not change throughout fetal development. Neuroendocrine cells were present throughout the entire period studied. Nonciliated cuboidal bronchiolar cells of the nonciliated population of the respiratory bronchiole appeared at 105 DGA (62% gestation) and matured in the postnatal period. We conclude that 1) although most of the differentiation of the lower airway occurs before birth, most of the cell types are not completely differentiated at birth; 2) the sequence of differentiation for the cells of the ciliated pseudostratified epithelial population is ciliated, nonciliated secretory, and basal; 3) the sequence of differentiation for the nonciliated secretory cell is similar to that of the secretory cells in more proximal airways; and 4) basal, neuroendocrine, and small mucous granule cells are not a part of the differentiation sequence of the other cell types.