Background: Although asthma is typically characterized as a childhood disease, it can develop later in life. Older asthmatic patients may be at increased risk for corticosteroid adverse effects. We developed a novel traditional Chinese medicine to treat asthma called antiasthma simplified herbal medicine intervention (ASHMI). Herbal products may offer safer adjunctive treatment for older asthmatic patients.Objective: To investigate the effects of ASHMI on characteristics of allergic asthma in an aged mouse model of asthma.Methods: BALB/c mice (6 weeks old [young] and 6, 12, and 18 months old [aged]) received ASHMI treatment before and during intraperitoneal ovalbumin sensitization and intratracheal challenges. The control groups were untreated, age-matched, ovalbumin-sensitized and ovalbumin-challenged mice (ovalbumin mice) and naive mice. After the final antigen challenge, airway pressure (defined as the time-integrated change in peak airway pressure) after acetylcholine provocation was measured, representing airway hyperresponsiveness, and bronchoalveolar lavage fluid, sera, lung tissues for histologic analysis, messenger RNA, and collagen were collected.Results: Mean time-integrated change in peak airway pressure values in 6-week-old and 6-, 12-, and 18-month-old ASHMI ovalbumin mice were significantly reduced compared with those of age-matched, nontreated ovalbumin mice. Bronchoalveolar lavage fluid eosinophil numbers were significantly lower in all ASHMI ovalbumin mice. Treatment with ASHMI of young and aged ovalbumin mice resulted in significantly decreased lung inflammation, detected via hematoxylin-eosin staining; airway mucous cell metaplasia, determined by means of periodic acid-Schiff staining; and messenger RNA copy numbers of the mucin gene MUC5AC. Levels of ovalbumin specific IgE and the T(H)2 cytokines interleukin 4 (IL-4), IL-5, and IL-13 in lung and splenocyte cultures were reduced. Interferon gamma secretion was increased. Treatment with ASHMI reduced collagen production.Conclusion: Treatment with ASHMI reduces several features of asthma in aged antigen-sensitized and antigen-challenged mice. Ann Allergy Asthma Immunol. 2010; 104:236-246.

A traditional Chinese Medicine (TCM) formula, Guo Min Kang (GMK), has been used in clinics in China for allergic diseases, including type I immediate hypersensitivity, a potentially fatal disease, but its modulatory mechanism remains elusive. The aim of this study was to investigate the modulatory mechanisms of GMK in a mouse model of Ag-induced anaphylaxis. Ag (conalbumin) sensitized mice were treated with either PBS (sham) or GMK before (schedule A) or during (schedule B) sensitization, and various anaphylactic parameters were measured following Ag challenge, including symptom score, cutaneous hypersensitivity response, mast cell degranulation, plasma histamine levels and the levels of specific IgE and T-cell responses. Systemic anaphylaxis was investigated in mice immediately following Ag challenge, and the results showed that GMK-treated mice from both treatment schedules A and B showed significantly reduced symptom scores when compared with the sham-treated group. The reduction in symptom score was associated with a significant reduction in the level of Ag-induced cutaneous immediate hypersensitivity. Also, GMK was able to suppress Ag-induced IgE production and T-cell responses, while it spares mitogen (Con A)-induced T-cell response. Further, treatment of mice with GMK abrogated the levels of Ag-induced histamine release and significantly reduced the number of degranulated mast cells. No effect of GMK was observed on the levels of total IgE and plasma histamine in naive mice. These results provide a basis for the modulation effect of GMK and suggest a potential utility of GMK as a prophylactic and therapeutic agent.

Background: Mucous hypersecretion increases asthma morbidity and mortality. Tumor necrosis factor alpha (TNF-alpha) levels are elevated in bronchoalveolar fluid, sputum, and monocyte membranes in some patients with asthma. Anti-TNF-alpha decreased asthma exacerbations and improved forced expiratory volume in I second in these patients. Whether anti-TNF-alpha reduces mucous cell metaplasia or hyperplasia has not been evaluated.Objective: To investigate the role of anti-TNF-alpha in mucous hypersecretion.Methods: BALB/c mice sensitized intraperitoneally and challenged intratracheally with ovalbumin were treated with 250 mu g of anti-TNF-alpha before ovalbumin sensitization and challenge or before only ovalbumin challenge. Control groups were sham treated. The tumor necrosis factor receptor (TNFR) mice (TNFR-/- and TNFR+/+) were identically sensitized and challenged. Seventy-two hours after the final challenge, the airway pressure time index (APTI), which measures airway hyperresponsiveness, was recorded. Mucous cell metaplasia was accessed by quantitative polymerase chain reaction for MUC-5AC (the epithelial cell mucous-inducing gene) and the percentage of periodic acid-Schiff (PAS) staining of bronchial epithelial cells. A human airway cell line (constitutively expressing MUC-5AC) was pretreated with a NF-kappa B inhibitor before TNF-alpha culture.Results: The mean (SE) fold change of MUC-5AC expression (compared with naive controls), the percentage of PAS-positive bronchiole epithelial cells, and the APTI decreased in BALB/c mice treated with anti-TNF-alpha before sensitization and challenge (4.9 [1.14], P = .007; 28.9% [6.8%], P < .001; and 545.8 [104.5] cm H2O/s, P < .001, respectively) and before challenge alone (9.3 [1.8], P = .03; 43.6% [10.7%], P = .009; and 896.8 [81.23] cm H2O/s, P = .06, respectively) compared with sham-treated mice (20.9 [3.9], 82.4% [1.8%], and 1,055 [30.6] cm H2O/s, respectively). MUC-5AC expression decreased in ovalbumin sensitized or challenged TNFR-/- (2.41 [0.4]) compared with ovalbumin sensitized or challenged TNFR+/+ mice (18.4 [2.5], P < .001). TNF-alpha-induced MUC-5AC expression in human airway culture significantly decreased with pretreatment of a NF-kappa B inhibitor.Conclusions: Anti-TNF-alpha treatment reduces airway mucous cell metaplasia in a mouse model of asthma, which may in part underlie its beneficial effect as asthma therapy. Ann Allergy Asthma Immunol. 2009; 103:295-303.

Background ImmuBalance™ is a koji fungus (Aspergillus oryzae) and lactic acid fermented soybean product. This unique production process is believed to create a food supplement that helps to induce or maintain normal immune response.

Leptin modulates energy metabolism and lung development. We hypothesize that the effects of leptin on postnatal lung development are volume dependent from 2 to 10 wk of age and are independent of hypometabolism associated with leptin deficiency. To test the hypotheses, effects of leptin deficiency on lung maturation were characterized in age groups of C57BL/6J mice with varying Lep ob genotypes. Quasi-static pressure-volume curves and respiratory impedance measurements were performed to profile differences in respiratory system mechanics. Morphometric analysis was conducted to estimate alveolar size and number. Oxygen consumption was measured to assess metabolic rate. Lung volume at 40-cmH2O airway pressure (V40) increased with age in each genotypic group, and V40 was significantly ( P < 0.05) lower in leptin-deficient ( ob/ ob) mice beginning at 2 wk. Differences were amplified through 7 wk of age relative to wild-type (+/+) mice. Morphometric analysis showed that alveolar surface area was lower in ob/ ob compared with +/+ and heterozygote ( ob/+) mice beginning at 2 wk. Unlike the other genotypic groups, alveolar size did not increase with age in ob/ ob mice. In another experiment, ob/ ob at 4 wk received leptin replacement (5 μg·g−1·day−1) for 8 days, and expression levels of the Col1a1, Col3a1, Col6a3, Mmp2, Tieg1, and Stat1 genes were significantly increased concomitantly with elevated V40. Leptin-induced increases in V40 corresponded with enlarged alveolar size and surface area. Gene expression suggested a remodeling event of lung parenchyma after exogenous leptin replacement. These data support the hypothesis that leptin is critical to postnatal lung remodeling, particularly related to increased V40 and enlarged alveolar surface area.

Background The effect of ageing on several pathologic features of allergic asthma (pulmonary inflammation, eosinophilia, mucus hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.Objective To evaluate lung inflammation, mucus metaplasia and AHR in relationship with age in murine models of allergic asthma comparing young and older mice.Methods Young (6 weeks) and older (6, 12, 18 months) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF), total inflammatory cell count and differential were measured. To evaluate mucus metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by quantitative PCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA. Results AHR developed in both aged and young OVA-sensitized/challenged mice (OVA mice), and was more significantly increased in young OVA mice than in aged OVA mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA mice than in young OVA mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA mice. All aged OVA mice had increased IL-5 and IFN-gamma mRNA expression in the lung and IL-5 and IFN-gamma protein levels from spleen cell cultures compared with young OVA mice. OVA-IgE was elevated to a greater extent in aged OVA mice.Conclusions Although pulmonary inflammation and mucus metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-gamma and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in ageing animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.

Previous studies from our laboratories showed lung development differences between inbred strains of mice. In the present study, the C57BL/6J (B6) and DBA/2J (D2) strains were examined for senescent-dependent differences with respect to the lung structure and function. Specifically, we hypothesize that senescent changes in lung vary between strains due to identifiable gene expression differences. Quasi-static pressure-volume curves and respiratory impedance measurements were performed on 2- and 20-mo-old B6 and D2 mice. Lung volume at 30 cm H(2)O (V(30)) pressure was significantly (P < 0.01) increased with age in both strains, but the increase was proportionally greater in D2 (68%) than in B6 (40%) mice. In addition, decreased elastic recoil pressure at 50% of V(30) and a reduction in airway resistance as a function of positive end-expiratory pressure were observed in 20-mo-old D2 mice but not in B6 mice. Morphometric analysis of lung parenchyma showed significant decreases in elastic fiber content with age in both strains, but the collagen content was significantly (P < 0.01) increased with age in D2 but not B6 mice at 20 mo. Furthermore, using quantitative RT-PCR methods, gene expression differences between strains suggested that D2 mice significantly (P < 0.05) downregulated the expressions of elastin (Eln) and procollagen I, III, and VI (Col1a1, Col3a1, and Col6a3) in lung tissue at 20 mo of age. These age-dependent changes were accompanied by an increased gene expression in matrix metalloproteinase 9 (Mmp9) in D2 and an increase in tissue inhibitor of matrix metalloproteinase (Timp1 and Timp4) in B6 mice. In conclusion, the results from the present study demonstrate that lung mechanics of both strains show significant age-dependent changes. However, changes in D2 mice are accelerated relative to B6 mice. Moreover, gene expression differences appear to be involved in the strain-specific changes of lung mechanic properties.

ABSTRACTEnhanced respiratory syncytial virus disease, a serious pulmonary disorder that affected recipients of an inactivated vaccine against respiratory syncytial virus in the 1960s, has delayed the development of vaccines against the virus. The enhanced disease was characterized by immune complex-mediated airway hyperreactivity and a severe pneumonia associated with pulmonary eosinophilia. In this paper, we show that complement factors contribute to enhanced-disease phenotypes. Mice with a targeted disruption of complement component C5 affected by the enhanced disease displayed enhanced airway reactivity, lung eosinophilia, and mucus production compared to wild-type mice and C5-deficient mice reconstituted with C5. C3aR expression in bronchial epithelial and smooth muscle cells in the lungs of C5-deficient mice was enhanced compared to that in wild-type and reconstituted rodents. Treatment of C5-deficient mice with a C3aR antagonist significantly attenuated airway reactivity, eosinophilia, and mucus production. These results indicate that C5 plays a crucial role in modulating the enhanced-disease phenotype, by affecting expression of C3aR in the lungs. These findings reveal a novel autoregulatory mechanism for the complement cascade that affects the innate and adaptive immune responses.

In the current study, we hypothesize that senescent-dependent changes between airway and lung parenchymal tissues of C57BL/6J (B6) mice are not synchronized with respect to altered lung mechanics. Furthermore, aging modifications in elastin fiber and collagen content of the airways and lung parenchyma are remodeling events that differ with time. To test these hypotheses, we performed quasi-static pressure-volume (PV) curves and impedance measurements of the respiratory system in 2-, 20-, and 26-mo-old B6 mice. From the PV curves, the lung volume at 30 cmH(2)O pressure (V(30)) and respiratory system compliance (Crs) were significantly (P < 0.01) increased between 2 and 20 mo of age, representing about 80-84% of the total increase that occurred between 2 and 26 mo of age. Senescent-dependent changes in tissue damping and tissue elastance were analogous to changes in V(30) and Crs; that is, a majority of the parenchymal alterations in the lung mechanics occurred between 2 and 20 mo of age. In contrast, significant decreases in airway resistance (R) occurred between 20 and 26 mo of age; that is, the decrease in R between 2 and 20 mo of age represented only 29% (P > 0.05) of total decrease occurring through 26 mo. Morphometric analysis of the elastic fiber content in lung parenchyma was significantly (P < 0.01) decreased between 2 and 20 mo of age. To the contrary, increased collagen content was significantly delayed until 26 mo of age (P < 0.01, 2 vs. 26 mo). In conclusion, our data demonstrate that senescent-dependent changes in airway and lung tissue mechanics are not synchronized in B6 mice. Moreover, the reduction in elastic fiber content with age is an early lung remodeling event, and the increased collagen content in the lung parenchyma occurs later in senescence.

Histochemical techniques for light and electron microscopy showed that metaphyseal osteoclasts in “incisors absent” rats contained greater than normal amounts of lysosomal acid phosphatase, aryl sulfatase and acid trimetaphosphatase. Lysosomal phosphatase activity at neutral pH was also elevated in the metaphyseal osteoclasts except in those cells immediately beneath the growth plate, where this enzyme was absent. The failure of any discernable resorption of organic matrix appeared to correlate with the absence of a ruffled border and a concomittant absence of extracellular lysosomal enzyme. Despite this failure, electron microscopic evidence of inorganic crystal removal was noted, suggesting that mineral dissolution represents a separate process from the enzymatic breakdown of organic matrix.

Cellular remodeling during angiogenesis in the lung is poorly described. Furthermore, it is the systemic vasculature of the lung and surrounding the lung that is proangiogenic when the pulmonary circulation becomes impaired. In a mouse model of chronic pulmonary thromboembolism, after left pulmonary artery ligation (LPAL), the intercostal vasculature, in proximity to the ischemic lung, proliferates and invades the lung (12). In the present study, we performed a detailed investigation of the kinetics of remodeling using histological sections of the left lung of C57Bl/6J mice after LPAL (4 h to 20 days) or after sham surgery. New vessels were seen within the thickened visceral pleura 4 days after LPAL predominantly in the upper portion of the left lung. Connections between new vessels within the pleura and pulmonary capillaries were clearly discerned by 7 days after LPAL. The visceral pleura and the lung parenchyma showed intense tissue remodeling, as evidenced by markedly elevated levels of both proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling positive cells. Rapidly dividing cells were predominantly macrophages and type II pneumocytes. The increased apoptotic activity was further quantified by caspase-3 activity, which showed a sixfold increase relative to naive lungs, by 24 h after LPAL. Because sham surgeries had little effect on measured parameters, we conclude that both thoracic wound healing and pulmonary ischemia are required for systemic neovascularization.

Background: Hypersecretion of mucus plays an important role in the pathogenesis and severity of asthma. The primary proteins in mucus are mucin glycoproteins; MUC-5AC is the primary airway mucin gene. The calcium chloride-activated channel gene hCLCA1 (gob-5 in the mouse) has been suggested to increase MUC-5AC gene expression, and both are increased in asthmatic patients and murine models. TNF-alpha increases the expression of these genes in vitro but has not been investigated in vivo.Objective: We sought to determine whether TNF-a increases gene expression of gob-5 and MUC-5AC and induces mucus cell metaplasia in vivo.Methods: Naive BALB/c mice received 50 ng of recombinant murine TNF-alpha (rmTNF-alpha) intratracheally daily for 1, 2, or 3 weeks; another group received the same dose of intratracheal rmTNF-alpha daily for 3 weeks and then alternate-day treatment for 3 additional weeks (total of 6 weeks). AKR mice received 50 ng of rmTNF-alpha intratracheally for 3 or 6 weeks daily. Naive nontreated mice were used as control animals. Airway gene products for gob-5 and MUC-5AC were determined by means of real-time PCR. Lung tissue sections were stained with periodic acid-Schiff/Alcian blue to assess mucus cell metaplasia.Results: rmTNF-alpha significantly increased gene expression of airway gob-5 and MUC-5AC after 2 weeks in the BALB/c mice. There was noticeable mucus staining in all mice treated for at least 3 weeks with TNF-a and in 80% of the mice receiving 2 weeks of treatment. After 3 weeks of treatment, the AKR mice also showed increased gob-5 expression.Conclusions: This study demonstrates for the first time that TNF-a alone in vivo is sufficient to increase airway mucus gene expression in 2 murine strains.

Objective: To discuss the potential use of the Chinese herbal formula MSSM-002 in treating asthma based on its effects on a murine model of allergic asthma, immunoregulatory actions on T(H)2 cells in vitro, and the means of standardization for herbal formula quality control.Data Sources: Information presented at the 2002 American College of Allergy, Asthma and Immunology (ACAAI) Annual Scientific Meeting International Symposium on Complementary Alternative Medicine in San Antonio, TX.Study Selection: All presentations from the ACAAI meeting that discussed MSSM-002 were considered for this review.Results: The Chinese herbal formula MSSM-002 suppressed airway hyperreactivity and eosinophilic inflammation in a murine model of allergic asthma. These effects were comparable to dexamethasone but were not accompanied by the suppression of T(H)1 responses seen with dexamethasone. In vitro studies demonstrated that MSSM-002 significantly decreased antigeninduced TH2 cytokine secretion by murine T(H)2 polarized splenocytes and human mucosal T(H)2 cell lines, which in contrast to dexamethasone did not cause apoptosis and was not cytotoxic but was associated with decreased GATA-3 expression. Chromatographic fingerprints of MSSM-002 and evaluation of in vivo actions showed that the quality of several batches of MSSM-002 was consistent.Conclusion: MSSM-002 has a therapeutic effect on allergic asthma and immunoregulatory actions on established T(H)2 cells and may prove to be of potential clinical benefit to asthma patients.

Rationale Ku-Shen (Sophora flavescens Ait) has been used for treating asthma in the practice of Traditional Chinese medicine (TCM) and Hawaiian herbal medicine. It is one of the major components in our anti-asthma herbal formula, MSSM-002. The purpose of this study was to investigate the pharmacological actions of Ku-Shen (KS) in a murine model of allergic asthma. Methods AKR/J mice (n=7-10 per group) were sensitized intraperitoneally twice and challenged 3 times intratracheally at 7-10 days intervals with conalbumin. KS was administered intragastrically 24 hours after the first challenge, and then twice daily for 17 days. The effects of KS on airway hyperreactivity (AHR), eosinophilic inflammation, IgE levels and Th2 production in vitro were determined. Results At the equivalent standard human adult dose, KS eliminated the development of AHR. Twice the usual dose (KS-2x) also significantly reduced airway inflammation as compared with the sham-treated group (13 ± 3 vs 43 ± 3%, p Conclusion KS suppressed allergic airway responses, reduced inflammation and down-regulated Th2 responses. These findings lend support to the anecdotal findings of Hawaiian and TCM practice that KS is of value in the treatment of asthma. Further studies are required to determine whether KS alone or with other herbs can be a useful alternative or complementary therapy in modern asthma treatment.

RATIONALE: The Chinese herbal medicine, Ling Zhi (LZ) has been used to treat asthma in traditional Chinese medical practice and is a major component in anti-asthma and food allergy herbal formulas.To explore the therapeutic mechanisms of LZ on allergic disorders, we compared the effects of LZ to dexamethasune (Dex) on Th2 polarized splenocytes in vitro.METHODS: Th2 polarized splenocytes were generated from spleens of AKR/J mice following conalbumin (CA) sensitization and challenge.Cells were cultured with CA in the presence or absence of LZ or Dex.Proliferation and cytokine profiles were determined.Cell viability and apoptosis were determined by trypan blue exclusion and TUNEL assays respectively.RESULTS: LZ treatment decreased Ag-induced proliferation (p<0.05vs untreated), and 1L-4 and IL-5 secretion (p<0.05vs untreated), and increased IFN-y secretion (p<0.05vs untreated) by Th2 polarized splenocytes at all tested doses.Dex treatment suppressed IFN-? as well as IL-4 and IL-5 cytokine secretion and proliferation.Furthermore, Dex-treated cultures contained few viable cells (5%), whereas the number of viable cells in LZ-treated cultures was essentially the same as in untreated cultures (60-70%).As expected, numerous cells underwent apoptosis in Dextreated cultures but only a small percent in LZ-treated cultures.CONCLUSIONS: LZ can suppress Th2 cell proliferation and switch established Th2 cells to Th0-1ike cells thereby re-establishing a Thl/Th2 balance.The actions of LZ on Th2 cells are different from Dex, which induced apoptosis and overall immunosuppression, The immunoregulato-:ry effect of LZ may be an important mechanism underlying its therapeutic effect on allergic disorders.

Decades of studies have shown the presence of neurotransmitters in the glomus cell, the putative chemosensory cell, and the involvement of neurotransmitters in carotid body chemotransmission (Gonzalez et al, 1994). We have been proposing that ACh is a major excitatory neurotransmitter in the cat carotid body (Fitzgerald, 2000). ACh fulfills most of criteria as an excitatory neurotransmitter in the carotid body. Regarding the presence of cholinergic receptors, our immunohistological study has shown that α7 subunits of nicotinic ACh receptors (nAChRs) locate at chemoreceptor nerve endings (Shirahata et al, 1998), and that α4 and β2 subunits of nAChRs in glomus cells (Ishizawa et al, 1994). Expression or localization of other subunits of nAChRs or subtypes of muscarinic ACh receptors (mAChRs) has not yet been known. Neuronal nAChRs are ligand-gated cation channels. They are composed of two a (a2-6) and three β (β2-4) subunits, or of five a (a7-9) subunits. Among variable combinations, α3β4, α4β2, and a7 types appear to be the major nAChRs, and they are widely but distinctively expressed in the nervous system (Lukas et al, 1999). Muscarinic AChRs are pharmacologically and molecular biologically classified into 5 subtypes (M1-M5). They are distributed in the nervous system, smooth muscles and gland (Caulfield and Birdsall, 1998). The purpose of the current study is to investigate the gene expression of major cholinergic receptors and to localize the receptor proteins in the cat chemosensitive unit (the carotid body and the petrosal ganglion).