Switching to Dupilumab from Other Biologics without a Treatment Interval in Patients with Severe Asthma: A Multi-Center Retrospective Study.

BACKGROUND: Dupilumab is a fully humanized monoclonal antibody that blocks interleukin-4 and interleukin-13 signals. Several large clinical trials have demonstrated the efficacy of dupilumab in patients with severe asthma. However, few studies have examined a switch to dupilumab from other biologics. METHODS: This retrospective, multi-center observational study was conducted by the Okayama Respiratory Disease Study Group. Consecutive patients with severe asthma who were switched to dupilumab from other biologics without a treatment interval between May 2019 and September 2021 were enrolled. Patients with a treatment interval of more than twice the standard dosing interval for the previous biologic prior to dupilumab administration were excluded. RESULTS: The median patient age of the 27 patients enrolled in this study was 57 years (IQR, 45-68 years). Eosinophilic chronic rhinosinusitis (ECRS)/chronic rhinosinusitis with nasal polyp (CRSwNP) was confirmed in 23 patients. Previous biologics consisted of omalizumab (n = 3), mepolizumab (n = 3), and benralizumab (n = 21). Dupilumab significantly improved FEV1 (median improvement: +145 mL) and the asthma control test score (median improvement: +2). The overall response rate in patients receiving dupilumab for asthma as determined using the Global Evaluations of Treatment Effectiveness (GETE) was 77.8%. There were no significant differences in the baseline characteristics of the GETE-improved group vs. the non-GETE-improved group. ECRS/CRSwNP improved in 20 of the 23 patients (87.0%). Overall, 8 of the 27 patients (29.6%) developed transient hypereosinophilia (>1500/µL), but all were asymptomatic and able to continue dupilumab therapy. CONCLUSIONS: Dupilumab was highly effective for the treatment of severe asthma and ECRS/CRSwNP, even in patients switched from other biologics without a treatment interval.

A retinoid X receptor partial agonist attenuates pulmonary emphysema and airway inflammation.

BACKGROUND: Retinoid X receptors (RXRs) are members of the nuclear receptor (NR) superfamily that mediate signalling by 9-cis retinoic acid, a vitamin A derivative. RXRs play key roles not only as homodimers but also as heterodimeric partners, e.g., for retinoic acid receptors, vitamin D receptors, and peroxisome proliferator-activated receptors. The NR family may also play important roles in the development of emphysema. However, the role of RXRs in the pathogenesis of emphysema is not well defined. METHODS: We developed a novel RXR partial agonist (NEt-4IB) and investigated its effect and mechanism compared to a full agonist (bexarotene) in a murine model of emphysema. For emphysema induction, BALB/c mice received intraperitoneal cigarette smoke extract (CSE) or intratracheal porcine pancreas elastase (PPE). Treatment with RXR agonists was initiated before or after emphysema induction. RESULTS: Treatment with NEt-4IB significantly suppressed the increase in static lung compliance and emphysematous changes in CSE-induced emphysema and PPE-induced established and progressive emphysema. NEt-4IB significantly suppressed PPE-induced neutrophilic airway inflammation and the levels of keratinocyte chemoattractant (KC), C-X-C motif ligand5 (CXCL5), interferon (IFN)-γ and IL-17. NEt-4IB also improved the matrix metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinase-1 (TIMP-1) imbalance and the reduced anti-oxidant activity in bronchoalveolar lavage (BAL) fluid. NEt-4IB suppressed PPE-induced vascular endothelial growth factor (VEGF) expression in the airway. Treatment with NEt-4IB and bexarotene significantly suppressed the increase in static lung compliance and emphysematous changes. However, adverse effects of RXR agonists, including hypertriglyceridemia and hepatomegaly, were observed in bexarotene-treated mice but not in NEt-4IB-treated mice. CONCLUSION: These data suggest that RXRs play crucial roles in emphysema and airway inflammation, and novel partial RXR agonists could be potential therapeutic strategies for the treatment of PPE- and CSE-induced emphysema.

Utility of a Fluorescence Microscopy Imaging System for Analyzing the DNA Ploidy of Pathological Megakaryocytes Including 5q- Syndrome.

To investigate megakaryocyte (MK) DNA ploidy in various hematological diseases, fluorescence microscopy imaging system (FMI) can be used to analyze DNA ploidy with cell morphology at the single-cell level by using specialized image-processing software. Here we compared DNA ploidy obtained by FMI measured with that obtained flow cytometry (FCM). With FMI, we could evaluate the DNA ploidy in long-term preserved bone marrow smear samples after staining. We next analyzed the MK DNA ploidy in 42 bone marrow smear samples including 26 myeloid neoplasm cases, and we compared the DNA ploidy and platelet counts in the patients' peripheral blood; the production of platelets was significantly high compared to DNA ploidy in the myeloproliferative neoplasms group. The FMI method revealed that the patients with 5q- syndrome exhibited relatively low DNA ploidy despite high platelet counts, and this result suggested that increased DNA ploidy is not indispensable to abundant platelet production. The FMI method for DNA ploidy will be a useful tool to clarify the relationship between DNA ploidy and platelet production by MKs.

Statistical description of the denatured structure of a single protein, staphylococcal nuclease, by FRET analysis.

Structural characterization of fully unfolded proteins is essential for understanding not only protein-folding mechanisms, but also the structures of intrinsically disordered proteins. Because an unfolded protein can assume all possible conformations, statistical descriptions of its structure are most appropriate. For this purpose, we applied Förster resonance energy transfer (FRET) analysis to fully unfolded staphylococcal nuclease. Artificial amino acids labeled with a FRET donor or acceptor were introduced by an amber codon and a four-base codon respectively. Eight double-labeled proteins were prepared, purified, and subjected to FRET analysis in 6 M urea. The observed behavior could be explained by a power law, R = αN0.44, where R, and N are the distance and the number of residues between donor and acceptor, and α is a coefficient. The index was smaller than the value expected for an excluded-volume random coil, 0.588, indicating that the fully unfolded proteins were more compact than polypeptides in good solvent. The FRET efficiency in the native state did not necessarily correlate to the distance obtained from crystal structure, suggesting that other factors such as the orientation factor made a substantial contribution to FRET.

Lavender Essential Oil and Its Main Constituents Inhibit the Expression of TNF-α-induced Cell Adhesion Molecules in Endothelial Cells.

Lavender essential oil (Lvn) has anti-inflammatory effects in an ovalbumin-sensitized murine model of asthma, and inhibits inflammatory cell infiltration into the lungs. The anti-inflammatory effects of Lvn on cell adhesion molecules are not clear. Here we evaluated the effects of Lvn and its main constituents, linalyl acetate (LA) and linalool (LO), on the expression of tumor necrosis factor-alpha (TNF-α)-induced cell adhesion molecules in murine brain endothelial bEnd.3 cells and human umbilical vein endothelial cells (HUVECs). The bEnd.3 cells were treated with Lvn, LA, or LO and subsequently stimulated with TNF-α. The mRNA expression levels of cell adhesion molecules were detected using RT-PCR. E-selectin and P-selectin protein and phosphorylated-NF-κB p65 were detected by western blotting. The effects of Lvn on HUVECs were measured by RT-PCR. In bEnd.3 cells, Lvn and LA suppressed TNF-α-induced E-selectin, P-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and phosphorylated-NF-κB p65 in the nucleus; LO did not suppress P-selectin or phosphorylated-NF-κB p65. Lvn inhibited TNF-α-induced E-selectin mRNA in HUVECs. These results indicate that Lvn and LA inhibit TNF-α-induced cell adhesion molecules in endothelial cells through the suppression of NF-κB activation. Consequently, Lvn or other essential oils including LA may be useful as alternative anti-inflammatory medicines.

Heat-induced native dimerization prevents amyloid formation by variable domain from immunoglobulin light-chain REI.

Amyloid light-chain (AL) amyloidosis is a protein-misfolding disease characterized by accumulation of immunoglobulin light chains (LCs) into amyloid fibrils. Dimerization of a full length or variable domain (VL ) of LC serves to stabilize the native state and prevent the formation of amyloid fibrils. We here analyzed the thermodynamic properties of dimerization and unfolding reactions by nonamyloidogenic VL from REI LC or its monomeric Y96K mutant using sedimentation velocity and circular dichroism. The data indicate that the equilibrium shifts to native dimerization for wild-type REI VL by elevating temperature due to the negative enthalpy change for dimer dissociation (-81.2 kJ·mol-1 ). The Y96K mutation did not affect the stability of the monomeric native state but increased amyloidogenicity. These results suggest that the heat-induced native homodimerization is the major factor preventing amyloid formation by wild-type REI VL . Heat-induced native oligomerization may be an efficient strategy to avoid the formation of misfolded aggregates particularly for thermostable proteins that are used at elevated temperatures under conditions where other proteins tend to misfold. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 5XP1 and 5XQY.

Contrasting roles for the receptor for advanced glycation end-products on structural cells in allergic airway inflammation vs. airway hyperresponsiveness.

The receptor for advanced glycation end-products (RAGE) is a multiligand receptor that belongs to the immunoglobulin superfamily. RAGE is reported to be involved in various inflammatory disorders; however, studies that address the role of RAGE in allergic airway disease are inconclusive. RAGE-sufficient (RAGE+/+) and RAGE-deficient (RAGE-/-) mice were sensitized to ovalbumin, and airway responses were monitored after ovalbumin challenge. RAGE-/- mice showed reduced eosinophilic inflammation and goblet cell metaplasia, lower T helper type 2 (Th2) cytokine production from spleen and peribronchial lymph node mononuclear cells, and lower numbers of group 2 innate lymphoid cells in the lung compared with RAGE+/+ mice following sensitization and challenge. Experiments using irradiated, chimeric mice showed that the mice expressing RAGE on radio-resistant structural cells but not hematopoietic cells developed allergic airway inflammation; however, the mice expressing RAGE on hematopoietic cells but not structural cells showed reduced airway inflammation. In contrast, absence of RAGE expression on structural cells enhanced innate airway hyperresponsiveness (AHR). In the absence of RAGE, increased interleukin (IL)-33 levels in the lung were detected, and blockade of IL-33 receptor ST2 suppressed innate AHR in RAGE-/- mice. These data identify the importance of RAGE expressed on lung structural cells in the development of allergic airway inflammation, T helper type 2 cell activation, and group 2 innate lymphoid cell accumulation in the airways. RAGE on lung structural cells also regulated innate AHR, likely through the IL-33-ST2 pathway. Thus manipulating RAGE represents a novel therapeutic target in controlling allergic airway responses.

Reactivity of CA19-9 and CA125 in histological subtypes of epithelial ovarian tumors and ovarian endometriosis.

Previous reports have shown that some ovarian endometrioid adenocarcinomas and ovarian clear cell adenocarcinomas derive from ovarian endometriosis (OE), and that endocervical-like mucinous borderline ovarian tumors are associated with OE. We examined the relationship between the staging and histological subtypes of OE or epithelial ovarian tumors (EOT) and the serum levels of carbohydrate antigen 19-9 (CA19-9) and carbohydrate antigen 125 (CA125) to evaluate the potential of these markers for preoperative diagnosis. First, we analyzed the preoperative serum levels of CA19-9 and CA125 in 195 patients who were histopathologically diagnosed with OE or EOT. We then performed a case-control study in which 308 women were enrolled, the 195 women described above and 113 healthy women as control subjects. Serum CA19-9 and CA125 levels were found to be useful in differentiating between OE and serous adenocarcinoma, but not between OE and other EOT. Moreover, serum CA19-9 levels were useful for preoperative assessment between OE and stage I mucinous borderline ovarian tumors, with or without the interstitial infiltration. In addition, considering that the serum CA19-9 levels in stage I mucinous borderline ovarian tumors were elevated via the interstitial infiltration of leukocytes and that precancerous lesions are associated with a cancerous glycosylation disorder in the process of inflammatory carcinogenesis, the CA19-9 level may be considered a suitable biomarker for estimating drug susceptibility.

Rigosertib induces cell death of a myelodysplastic syndrome-derived cell line by DNA damage-induced G2/M arrest.

A multi-kinase inhibitor, rigosertib (ON 01910.Na) has recently been highlighted as a novel type of anti-cancer agent for the treatment of the myelodysplastic syndromes (MDS), but its action mechanisms remain to be clarified. We investigated the in vitro effects of rigosertib on an MDS-derived cell line MDS-L and a myeloid leukemia cell line HL-60. Rigosertib suppressed the proliferation of both HL-60 and MDS-L cells and induced apoptosis by inhibition of the PI3 kinase/Akt pathway. As the effects on cell cycle, rigosertib treatment promoted the phosphorylation of histone H2AX and led to the DNA damage-induced G2/M arrest. In addition, an immunofluorescence staining study demonstrated the abnormal localization of aurora A kinase, suggesting that rigosertib causes perturbation of spindle assembly and deregulated mitotic patterns towards cell cycle arrest and apoptosis. We also found that rigosertib exerted growth inhibitory effects on two lymphoid cell lines, Jurkat and Ramos. We further examined the molecular pathways influenced by rigosertib from the gene expression profiling data of MDS-L cells and found a possible involvement of rigosertib treatment in the upregulation of the genes related to microtubule kinetics and the downregulation of the mRNA degradation system. The gene set enrichment analysis showed the suppression of "nonsense-mediated mRNA decay (NMD)" as the most significantly affected gene set. These data provide a new aspect and a potential utility of rigosertib for the treatment of refractory hematopoietic malignancies.

Decreased amyloidogenicity caused by mutational modulation of surface properties of the immunoglobulin light chain BRE variable domain.

Amyloid formation by immunoglobulin light chain (LC) proteins is associated with amyloid light chain (AL) amyloidosis. Destabilization of the native state of the variable domain of the LC (VL) is known to be one of the critical factors in promoting the formation of amyloid fibrils. However, determining the key residues involved in this destabilization remains challenging, because of the existence of a number of intrinsic sequence variations within VL. In this study, we identified the key residues for destabilization of the native state of amyloidogenic VL in the LC of BRE by analyzing the stability of chimeric mutants of BRE and REI VL; the latter immunoglobulin is not associated with AL amyloidosis. The results suggest that the surface-exposed residues N45 and D50 are the key residues in the destabilization of the native state of BRE VL. Point mutations at the corresponding residues in REI VL (K45N, E50D, and K45N/E50D) destabilized the native state and increased amyloidogenicity. However, the reverse mutations in BRE VL (N45K, D50E, and N45K/D50E) re-established the native state and decreased amyloidogenicity. Thus, analyses using chimeras and point mutants successfully elucidated the key residues involved in BRE VL destabilization and increased amyloidogenic propensity. These results also suggest that the modulation of surface properties of wild-type VL may improve their stability and prevent the formation of amyloid fibrils.

Lavender essential oil inhalation suppresses allergic airway inflammation and mucous cell hyperplasia in a murine model of asthma.

AIMS: Lavender essential oil (Lvn) has been reported to have anti-inflammatory effects. Bronchial asthma is characterized by bronchial allergic inflammation with airway remodeling. Therefore, we evaluated the anti-inflammatory effect of Lvn on experimentally induced bronchial asthma in a murine model. MAIN METHODS: BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA) at days 0 and 14, and subsequently challenged with nebulized OVA on days 28-30 (Control-Asthma group). Mice in the treatment group inhaled Lvn on days 14-31 (Lvn-Asthma group). The allergic inflammatory response was determined on days 32 and 33. KEY FINDINGS: An increase in airway resistance was inhibited in the Lvn-Asthma group than in the Control-Asthma group. The Lvn-Asthma group showed lower total cell numbers and eosinophils in bronchoalveolar lavage (BAL) fluids and peribronchial and perivascular tissues when compared with the Control-Asthma group. The Lvn-Asthma group also had less mucin hyperplasia than the Control-Asthma group. Furthermore, the Lvn-Asthma group showed lower interleukin (IL)-5 and IL-13 cytokine levels in BAL fluids, as well as reduced IL-4 and IL-5 mRNA expression in lung tissue, compared with the Control-Asthma group and determined by FlowCytomix and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. In addition, Lvn inhalation reduced Muc5b mRNA expression in the lungs without significantly changing the expression of Muc5ac mRNA. SIGNIFICANCE: Lvn inhibits allergic inflammation and mucous cell hyperplasia with suppression of T-helper-2 cell cytokines and Muc5b expression in a murine model of asthma. Consequently, Lvn may be useful as an alternative medicine for bronchial asthma.

Effect of a cysteinyl leukotriene receptor antagonist on experimental emphysema and asthma combined with emphysema.

The incidence of overlapping bronchial asthma and chronic obstructive pulmonary disease has increased in recent years. Cysteinyl leukotrienes (CysLTs) play an important role in asthma, and the type 1 CysLT receptor (CysLT1R) is expressed by many inflammatory cells. We evaluated the effect of montelukast, a CysLT1R antagonist, on mouse models of asthma, porcine pancreatic elastase (PPE)-induced emphysema, and asthma combined with emphysema. Mice were sensitized with ovalbumin (OVA) on Days 0 and 14 and subsequently challenged with OVA on Days 28, 29, and 30. Pulmonary emphysema was induced by intratracheal instillation of PPE on Day 25. Mice were treated subcutaneously with montelukast or vehicle from Day 25 to Day 31. Airway hyperresponsiveness (AHR), static compliance; the number of inflammatory cells, the levels of cytokines, chemokines, LTs, and perforin in the bronchoalveolar lavage fluid, and the quantitative morphometry of lung sections were analyzed on Day 32. Treatment with montelukast significantly attenuated the AHR and eosinophilic airway inflammation in OVA-sensitized and OVA-challenged mice. Administration of montelukast significantly reduced the AHR, static compliance, and neutrophilic airway inflammation, while attenuating emphysematous lung changes, in PPE-treated mice. In PPE-treated mice subjected to allergen sensitization and challenges, montelukast significantly suppressed the AHR, static compliance, and eosinophilic and neutrophilic airway inflammation in addition to the development of experimentally induced emphysema in the lungs. Our data suggest that CysLT1R antagonists may be effective in ameliorating the consequences of PPE-induced lung damage and the changes that follow allergen sensitization and challenges.

Molecular dissection of IZUMO1, a sperm protein essential for sperm-egg fusion.

Although the membrane fusion of spermatozoon and egg cells is the central event of fertilization, the underlying molecular mechanism remains virtually unknown. Gene disruption studies have showed that IZUMO1 on spermatozoon and CD9 on oocyte are essential transmembrane proteins in sperm-egg fusion. In this study, we dissected IZUMO1 protein to determine the domains that were required for the function of sperm-egg fusion. We found that a fragment of the N terminus (Asp5 to Leu113) interacts with fertilization inhibitory antibodies. It also binds to the egg surface and effectively inhibits fusion in vitro. We named this fragment 'IZUMO1 putative functional fragment (IZUMO1PFF)'. Surprisingly, IZUMO1PPF still maintains binding ability on the egg surface of Cd9(-/-) eggs. A series of biophysical measurements using circular dichroism, sedimentation equilibrium and small angle X-ray scattering revealed that IZUMO1PFF is composed of an N-terminal unfolded structure and a C-terminal ellipsoidal helix dimer. Egg binding and fusion inhibition were not observed in the IZUMO1PFF derivative, which was incapable of helix formation. These findings suggest that the formation of a helical dimer at the N-terminal region of IZUMO1 is required for its function. Cos-7 cells expressing the whole IZUMO1 molecule bound to eggs, and IZUMO1 accumulated at the interface between the two cells, but fusion was not observed. These observations suggest that IZUMO1 alone cannot promote sperm-egg membrane fusion, but it works as a factor that is related to the cellular surface interaction, such as the tethering of the membranes by a helical region corresponding to IZUMO1PFF-core.

Effect of fudosteine, a cysteine derivative, on airway hyperresponsiveness, inflammation, and remodeling in a murine model of asthma.

AIMS: Fudosteine is a cysteine derivative that is used as an expectorant in chronic bronchial inflammatory disorders. It has been shown to decrease the number of goblet cells in an animal model. This study examined the effects of fudosteine on airway inflammation and remodeling in a murine model of chronic asthma. MAIN METHODS: BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA), and subsequently challenged with nebulized ovalbumin three days a week for four weeks. Seventy-two hours after the fourth challenge, airway hyperresponsiveness (AHR) and the cell composition of bronchoalveolar lavage (BAL) fluid were assessed. Fudosteine was administered orally at 10mg/kg or 100mg/kg body weight from the first to the fourth challenge. KEY FINDINGS: We investigated the effects of fudosteine on the development of allergic airway inflammation and airway hyperresponsiveness after chronic allergen challenges. The administration of fudosteine during the challenge with ovalbumin prevented the development of airway hyperresponsiveness and accumulation of lymphocytes in the airways. Eotaxin, IL-4, and TGF-ß levels and the relative intensity of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9) in BAL fluid were reduced by the fudosteine treatment; however, the number of eosinophils in BAL fluid and serum IgE levels did not change. The expression of TGF-ß, the development of goblet cell hyperplasia, subepithelial collagenization, and basement membrane thickening were also reduced by the fudosteine treatment. SIGNIFICANCE: These results indicate that fudosteine is effective in reducing airway hyperresponsiveness, airway inflammation, and airway remodeling in a murine model of chronic asthma.

Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses.

BACKGROUND: Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice. METHODS: BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge. RESULTS: Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-ß1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice. CONCLUSION: These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.

Blocking the leukotriene B4 receptor 1 inhibits late-phase airway responses in established disease.

Most of the studies investigating the effectiveness of blocking the leukotriene B4 (LTB4) receptor 1 (BLT1) have been performed in models of primary or acute allergen challenge. The role of the LTB4-BLT1 pathway in secondary challenge models, where airway hyperresponsiveness (AHR) and airway inflammation have been established, has not been defined. We investigated the effects of blocking BLT1 on early- and late-phase development of AHR and airway inflammation in previously sensitized and challenged mice. Female BALB/c mice were sensitized (Days 1 and 14) and challenged (primary, Days 28-30) with ovalbumin. On Day 72, mice were challenged (secondary) with a single OVA aerosol, and the early and late phases of AHR and inflammation were determined. Specific blockade of BLT1 was attained by oral administration of a BLT1 antagonist on Days 70 through 72. Administration of the antagonist inhibited the secondary ovalbumin challenge-induced alterations in airway responses during the late phase but not during the early phase, as demonstrated by decreases in AHR and in bronchoalveolar lavage neutrophilia and eosinophilia 6 and 48 hours after secondary challenge. The latter was associated with decreased levels of KC protein, macrophage inflammatory protein 2, and IL-17 in the airways. These data identify the importance of the LTB4-BLT1 pathway in the development of late-phase, allergen-induced airway responsiveness after secondary airway challenge in mice with established airway disease.

Experimental pulmonary granuloma mimicking sarcoidosis induced by Propionibacterium acnes in mice.

Propionibacterium acnes has been implicated as an etiologic agent of sarcoidosis since the isolation of this bacterium from sarcoid lesions. We experimentally produced a murine pulmonary granuloma model using P. acnes with several features that simulate sarcoidosis. Mice were sensitized with heat-killed P. acnes and complete Freund's adjuvant and were subsequently challenged with heat-killed P. acnes at 2-week intervals. P. acnes-challenged mice developed epitheloid cell granulomas in the lungs. These mice showed a pulmonary immune response characterized by an increased number of T-lymphocytes, especially CD4+ cells, and the ratio of CD4+/CD8+ in bronchoalveolar lavage (BAL) fluid also increased. Furthermore, significant elevations in both angiotensin-converting enzyme (ACE) serum levels and antibody titers against P. acnes were observed. Mice sensitized with P. acnes without complete Freund's adjuvant were capable of forming pulmonary granulomas, which appeared to be caused by indigenous P. acnes. The genome of P. acnes was found in the lungs, BAL cells, hilar lymph nodes, liver, and spleen in non-sensitized mice, which were thought to be germ-free. These results suggest that the immune response against indigenous P. acnes may play an important role in the pathogenesis of granuloma formation in a murine model.

Nonlocal interactions are responsible for tertiary structure formation in staphylococcal nuclease.

Rapid molecular collapse mediated by nonlocal interactions is believed to be a crucial event for protein folding. To investigate the role of nonlocal interactions in tertiary structure formation, we performed a nonlocal interaction substitution mutation analysis on staphylococcal nuclease (SNase). Y54 and I139 of wild-type (WT) SNase and Delta140-149 were substituted by cysteine to form intramolecular disulfide bonds, respectively called WT-SS and Delta140-149-SS. Under physiological conditions, the reduced form of Delta140-149-SS appears to assume a denatured structure; in contrast, the oxidized form of Delta140-149-SS forms a native-like structure. From this result, we conclude that the C-terminal region participates in a nonlocal interaction that is indispensable for the native structure. Although the oxidized form of WT-SS assumes a more compact denatured structure under acidic conditions than the WT, the kinetic measurements reveal that the refolding reactions of both the reduced and oxidized forms of WT-SS are similar to those of the WT, suggesting that an intact nonlocal interaction is established within the dead time (22 ms). On the basis of these results, we propose that the native nonlocal contact established at the early stage of the folding process facilitates further secondary structure formation.

Nucleotide-dependent conformational changes and assembly of the AAA ATPase SKD1/VPS4B.

SKD1/VPS4B belongs to the adenosine triphosphatases associated with diverse cellular activities (AAA) family and regulates multivesicular body (MVB) biogenesis. SKD1 changes its oligomeric state during the ATPase cycle and subsequently releases endosomal sorting complex required for transport (ESCRT) complexes from endosomes during the formation of MVBs. In this study, we describe domain motions in monomeric SKD1 on ATP and ADP binding. Nucleotides bind between the alpha/beta and the alpha-helical domains of SKD1, inducing a approximately 20 degrees domain rotation and closure of the binding site, which are similar to the changes observed in the AAA+ ATPase, HslU. Gel filtration and small-angle X-ray scattering experiments showed that the ATP-bound form of SKD1 oligomerizes in solution, whereas ADP-bound and apo forms of SKD1 exist as monomers, even though the conformations of the ADP- and ATP-bound forms are nearly identical. Nucleotide-bound SKD1 structures are compatible with a hexameric ring arrangement reminiscent of the AAA ATPase p97 D1 ring. In the hexameric ring model of SKD1, Arg290 from a neighboring molecule binds to the gamma-phosphate of ATP, which promotes oligomerization of the ATP-bound form. ATP hydrolysis would eliminate this interaction and subsequent nucleotide release causes the domains to rotate, which together lead to the disassembly of the SKD1 oligomer.

Inflammatory markers in exhaled breath condensate from patients with asthma.

BACKGROUND AND OBJECTIVE: Evaluation of airway inflammation is important for the diagnosis and treatment of asthma. Exhaled breath condensate (EBC) is a minimally invasive method for assessing inflammation and may be useful for monitoring airway inflammation in asthma. The aims of this study were to establish an EBC collection method, to assess biomarkers reflecting asthmatic airway inflammation, and to determine the relationship of these biomarkers with asthma severity and lung function. METHODS: Fifty-eight non-smoking healthy subjects, seven asymptomatic smokers, nine subjects with common cold and 55 asthmatics with disease severity ranging from mild intermittent to severe persistent were studied. The efficacy of a pipette method was compared with that of a commercial collecting device. pH, CRP, albumin, hydrogen peroxide (H(2)O(2)) and nitrite/nitrate levels were measured in EBC. RESULTS: Except for the quantity of EBC collected and albumin levels, there were no differences between the commercial method and the pipette method in levels of biomarkers measured. Levels of CRP, H(2)O(2) and nitrite/nitrate were significantly higher in the asthma group than that in the control group. In terms of asthma severity, pH and levels of CRP, H(2)O(2) and nitrate were significantly higher in the mild persistent group than that in the other groups. In addition, H(2)O(2) levels in EBC correlated significantly with the level of nitrite/nitrate. FEV(1) and PEF showed significant negative correlations with H(2)O(2) and nitrite/nitrate levels. CONCLUSION: Measurement of EBC biomarkers is a non-invasive and useful way to evaluate airway inflammation in patients with asthma.