Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor.

The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs.

Pasteurella multocida toxin prevents osteoblast differentiation by transactivation of the MAP-kinase cascade via the Gα(q/11)--p63RhoGEF--RhoA axis.

The 146-kDa Pasteurella multocida toxin (PMT) is the main virulence factor to induce P. multocida-associated progressive atrophic rhinitis in various animals. PMT leads to a destruction of nasal turbinate bones implicating an effect of the toxin on osteoblasts and/or osteoclasts. The toxin induces constitutive activation of Gα proteins of the G(q/11)-, G12/13- and G(i)-family by deamidating an essential glutamine residue. To study the PMT effect on bone cells, we used primary osteoblasts derived from rat calvariae and stromal ST-2 cells as differentiation model. As marker of functional osteoblasts the expression and activity of alkaline phosphatase, formation of mineralization nodules or expression of specific transcription factors as osterix was determined. Here, we show that the toxin inhibits differentiation and/or function of osteoblasts by activation of Gα(q/11). Subsequently, Gα(q/11) activates RhoA via p63RhoGEF, which specifically interacts with Gα(q/11) but not with other G proteins like Gα12/13 and Gα(i). Activated RhoA transactivates the mitogen-activated protein (MAP) kinase cascade via Rho kinase, involving Ras, MEK and ERK, resulting in inhibition of osteoblast differentiation. PMT-induced inhibition of differentiation was selective for the osteoblast lineage as adipocyte-like differentiation of ST-2 cells was not hampered. The present work provides novel insights, how the bacterial toxin PMT can control osteoblastic development by activating heterotrimeric G proteins of the Gα(q/11)-family and is a molecular pathogenetic basis for understanding the role of the toxin in bone loss during progressive atrophic rhinitis induced by Pasteurella multocida.

Swine atrophic rhinitis caused by pasteurella multocida toxin and bordetella dermonecrotic toxin.

Atrophic rhinitis is a widespread and economically important swine disease caused by Pasteurella multocida and Bordetella bronchiseptica. The disease is characterized by atrophy of the nasal turbinate bones, which results in a shortened and deformed snout in severe cases. P. multocida toxin and B. bronchiseptica dermonecrotic toxin have been considered to independently or cooperatively disturb the osteogenesis of the turbinate bone by inhibiting osteoblastic differentiation and/or stimulating bone resorption by osteoclasts. Recently, the intracellular targets and molecular actions of both toxins have been clarified, enabling speculation on the intracellular signals leading to the inhibition of osteogenesis.

Molecular biology of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is the causative agent of progressive atrophic rhinitis in swine. The 146 kDa single-chain toxin harbours discrete domains important for receptor binding, internalisation and biological activity. The molecular basis of the toxin's activity is the deamidation of a specific glutamine residue in the α-subunit of heterotrimeric G proteins. This results in an inhibition of the inherent GTPase activity leading to a constitutively active phenotype of the G protein. Due to the ability of the toxin to act on various families of heterotrimeric G proteins, a large subset of signal transduction pathways is stimulated.

Association of Bordetella dermonecrotic toxin with the extracellular matrix.

BACKGROUND: Bordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown. RESULTS: Fluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active. CONCLUSIONS: Components in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

Interindividual variability in nasal filtration as a function of nasal cavity geometry.

BACKGROUND: Interindividual variability in nasal filtration is significant due to interindividual differences in nasal anatomy and breathing rate. Two important consequences arise from this variation among humans. First, devices for nasal drug delivery may furnish quite different doses in the nasal passages of different individuals, leading to different responses to therapeutic treatment. Second, people with poor nasal filtration may be more susceptible to adverse health effects when exposed to airborne particulate matter (PM) due to greater lung deposition. Although interindividual variability of nasal filtration has been reported by several authors, a relationship for predicting filtration efficiency from nasal anatomy and ventilation is still lacking. Such a relationship is needed to (1) devise nasal drug delivery systems and (2) define limits of exposure to PM that are effective for the human population at large. METHODS: Anatomically correct nasal replicas of five adults (four healthy individuals and one atrophic rhinitis patient) were used in aerosol experiments to measure nasal deposition of 1-12-microm particles. The dependence of nasal filtration on nasal anatomy and breathing rate was investigated using various definitions of the Stokes number as well as phenomenological Impaction Parameters proposed in the literature. RESULTS: Interindividual variability among the healthy adults was nearly eliminated when nasal filtration was plotted against a specific definition of the Stokes number or against a pressure-based Impaction Parameter. Nasal filtration in the atrophic rhinitis patient was lower than in the healthy subjects. CONCLUSIONS: The new definition of the Stokes number introduced in this study, which is based on a new definition of the characteristic diameter of the nasal passages, nearly eliminated interindividual differences in nasal filtration. Our results suggest that it is possible to estimate nasal filtering efficiency using measurements of transnasal pressure drop.

Study of surfactant level in cases of primary atrophic rhinitis.

The surfactant system of the nose was examined biochemically in control cases and compared to cases of primary atrophic rhinitis. The study group included 25 cases with primary atrophic rhinitis compared to 10 normal volunteers. Biochemical analysis of the nasal aspirate in these cases revealed the presence of phospholipids constituting surfactant with phosphatidylcholine constituting 75.35 per cent of the total phospholipids. Biochemical analysis of the nasal aspirate in cases with primary atrophic rhinitis revealed a significant decrease in the total phospholipids compared to normal cases and also a significant change in the phospholipid profile. Thus significant biochemical changes in the surfactant system of the nose is an evident and early finding in cases of primary atrophic rhinitis. This suggests a possible role for surfactant deficiency in the aetiopathogenesis of cases of primary atrophic rhinitis.

Histochemical and lectinhistochemical studies on nasal mucosa of pigs with or without respiratory diseases.

Histochemical and lectinhistochemical examinations were carried out on nasal mucosa of pigs with or without respiratory diseases. As the results, both acid and neutral mucins coexisted in nasal mucosa of normal pigs while acid sialomucins were mainly observed in nasal mucosa of pigs infected with Bordetella bronchiseptica and/or Pasteurella multocida. Lectinhistochemistry revealed that the nasal epithelial cells of normal pigs were rich in N-acetylgalactosamine, fucose and N-acetyl-glucosamine residues which showed a tendency to disappear in porcine cytomegalovirus infection and to increase in atrophic rhinitis, respectively.

Effects of ammonia inhalation and acetic acid pretreatment on colonization kinetics of toxigenic Pasteurella multocida within upper respiratory tracts of swine.

Pigs reared in intensive production systems are continuously exposed to ammonia released by the microbial degradation of their excrement. Exposure to this gas has been shown to increase the severity of the disease progressive atrophic rhinitis by facilitating colonization of the pig's upper respiratory tract by Pasteurella multocida. The etiological mechanism responsible for this synergy was investigated by studying the colonization kinetics of P. multocida enhanced by ammonia and comparing them with those evoked by an established disease model. Three-week-old Large White piglets were weaned and allocated to five experimental groups (groups A to E). Pigs in groups A and B were exposed continuously to ammonia at 20 ppm for the first 2 weeks of the study. Pigs in group C were pretreated with 0.5 ml of 1% acetic acid per nostril on days -2 and -1 of the study. On day 0 all the pigs in groups A, C, and D were inoculated with 1.4 x 10(8) toxigenic P. multocida organisms given by the intranasal route. The kinetics of P. multocida colonization were established by testing samples obtained at weekly intervals throughout the study. The study was terminated on day 37, and the extent of turbinate atrophy was determined by using a morphometric index. The results of the study showed that exposure to aerial ammonia for a limited period had a marked effect on the colonization of toxigenic P. multocida in the nasal cavities of pigs, which resulted in the almost total exclusion of commensal flora. In contrast, ammonia had only a limited effect on P. multocida colonization at the tonsil. The exacerbation of P. multocida colonization by ammonia was restricted to the period of ammonia exposure, and the number of P. multocida organisms colonizing the upper respiratory tract declined rapidly upon the cessation of exposure to ammonia. During the exposure period, the ammonia levels in mucus recovered from the nasal cavity and tonsil were found to be 7- and 3.5-fold higher, respectively, than the levels in samples taken from unexposed controls. Acetic acid pretreatment also induced marked colonization of the nasal cavity which, in contrast to that induced by ammonia, persisted throughout the time course of the study. Furthermore, acetic acid pretreatment induced marked but transient colonization of the tonsil. These findings suggest that the synergistic effect of ammonia acts through an etiological mechanism different from that evoked by acetic acid pretreatment. A strong correlation was found between the numbers of P. multocida organisms isolated from the nasal cavity and the severity of clinical lesions, as determined by using a morphometric index. The data presented in the paper highlight the potential importance of ammonia as an exacerbating factor in respiratory disease of intensively reared livestock.

Delayed eosinophil programmed cell death in vitro: a common feature of inhalant allergy and extrinsic and intrinsic atopic dermatitis.

The present studies were undertaken to characterize the potential role of eosinophil programmed cell death (PCD) in atopic diseases. Peripheral blood eosinophil PCD was found to be delayed in inhalant allergy (p < 0.05) and delayed to an even greater extent in atopic dermatitis (AD) (p < 0.0001) when compared to nonatopic subjects. There was no difference in the occurrence of PCD between the extrinsic and the intrinsic type of AD, pointing to a secondary role of specific sensitization. Blockade of eosinophil PCD was not responsible for peripheral blood eosinophilia, because we found no obvious relationship of eosinophil survival to blood eosinophil count. Eosinophil supernatants of more patients with AD than of patients with inhalant allergy dose-dependently inhibited PCD in nonatopic eosinophils, and it was shown that this effect was possibly due to autocrine production of granulocyte-macrophage-colony stimulating factor, probably IL-5. Eosinophil expression of CD95 (Fas antigen) did not change over time in culture and was not modulated by cytokines prolonging eosinophil survival. In contrast, IL-3, IL-5, and granulocyte-macrophage-colony stimulating factor caused an upregulated expression of CD69. However, in AD, CD69 on eosinophils was upregulated without the need of exogenous growth factor or factors over time in culture, thus confirming an autocrine production of proeosinophilic cytokines. In conclusion, our data clearly indicate that eosinophil PCD is markedly delayed in the so-called atopic diseases irrespective of allergen sensitization and suggest that this effect is mediated by the autocrine production of growth factors by eosinophils.

A suggested mechanism for the pathogenesis of infectious atrophic rhinitis.

Boivin extracts of Bordetella bronchiseptica inhibited or uncoupled the energized processes of bovine heart and pig heart mitochondria. Energy-dependent accumulation of calcium phosphate by both types of mitochondria was markedly inhibited by the extracts. The thesis is advanced that Boivin extracts of B. bronchiseptica may contain the membrane-damaging component responsible for infectious atrophic rhinitis. The action of the extracts was decreased by heat but seemed rather insensitive to digestion by trypsin or extraction by lipid solvents. Attempts to resolve the active component from the extracts were unsuccessful.