Inhibitory effect of Lactobacillus gasseri TMC0356 and Lactobacillus GG on enhanced vascular permeability of nasal mucosa in experimental allergic rhinitis of rats

The nasal vascular permeability of ovablumin (OVA)-sensitized Brown Norway rats was evaluated by analyzing a brilliant blue concentration in perfusate from the nose after exposure of the nasal mucus to OVA. Oral administration of Lactobacillus GG and L. gasseri TMC0356 significantly inhibited the increase in nasal vascular permeability (P<0.01). The serum IgE of the tested rats also decreased, although the change was not statistically significant. These results indicate that Lactobacillus GG and L. gasseri TMC0356 might alleviate nasal allergic symptoms by suppressing the increase in nasal vascular permeability caused by local inflammation associated with allergic rhnititis.     

Preliminary human study for possible alteration of serum immunoglobulin E production in perennial allergic rhinitis with fermented milk prepared with Lactobacillus gasseri TMC0356

The fermented milk prepared with Lactobacillus gasseri TMC0356 was administered at 200 ml per day for 4 weeks to 15 subjects with high serum IgE levels and perennial allergic rhinitis. The serum total IgE concentration was significantly reduced after 28 days' exposure to the fermented milk (P <0.05) compared to that before the intervention. The serum IgE specific to Acari and those to Japanese cedar pollen also significantly declined (P <0.05). T helper 1 (Th1) cells in the composition of their peripheral blood mononuclear cells (PBMCs) significantly increased after 14 days (P <0.01) and after 28 days (P <0.05). These results suggest that the fermented milk prepared with L. gasseri TMC0356 may alter serum IgE concentration, at least partly by enhancement of Th1 immune responses of the subjects with high concentration of serum IgE. However, further studies are still necessary to know the underlying mechanisms by which the tested fermented milk could influence the host immunity.     

Differentiated implication of Lactobacillus GG and L. gasseri TMC0356 to immune responses of murine Peyer's patch

Lactobacillus GG and L. gasseri TMC0356 were examined for their potential to alter the immune responses of murine PP cells in vitro and in vivo. Lactobacillus GG and L. gasseri TMC0356 characteristically stimulated the production of IL-12, IL-6, IFN-γ and IgA from isolated PP cells in vitro . Anatomical analysis indicated uptake of these bacteria by the PP tissue after giving orally in mice. Isolated PP cells exposed to Lactobacillus GG in vivo secreted more IFN-γ, IL-6 and total IgA, whereas those exposed to L. gasseri TMC0356 in vivo did not exhibit altered immune responses in terms of cytokine and IgA production. Therefore, these two bacteria might exhibit different immunodulatory effects in host animals by strain-dependent interaction with gut-associated lymphoid tissues in vivo .     

Orally administered heat-killed Lactobacillus gasseri TMC0356 alters respiratory immune responses and intestinal microbiota of diet-induced obese mice

Aims: To investigate the influence of heat‐killed Lactobacillus gasseri TMC0356 on changes in respiratory immune function and intestinal microbiota in a diet‐induced obese mouse model. Methods and Results: Male C57BL/6J mice were fed a high‐fat diet for 16 weeks. After 8 weeks, the high‐fat‐diet‐induced obese mice (DIO mice) were randomly divided into two 0067roups, the DIO and DIO0356 groups. DIO0356 group mice were orally fed with heat‐killed TMC0356 every day for 8 weeks, while DIO group mice were exposed to 0·85% NaCl over the same time period as controls. After intervention, the pulmonary mRNA expression of cytokines and other immune molecules in DIO0356 mice compared to those in DIO group mice was significantly increased (P < 0·05, P < 0·01). In faecal bacterial profiles, analysed using the terminal restriction fragment length polymorphism (T‐RFLP) method, T‐RFLP patterns in 75% of the DIO0356 group mice were apparently changed compared with those in control group mice. Conclusion: These results suggest that inactive lactobacilli may stimulate the respiratory immune responses of obese host animals to enhance their natural defences against respiratory infection, partially associating with their potent impact on intestinal microbiota. Significance and Impact of the Study: We have demonstrated that oral administration of inactive lactobacilli may protect host animals from the lung immune dysfunction caused by obesity.     

Enhancement of immunoregulatory effects of Lactobacillus gasseri TMC0356 by heat treatment and culture medium

Aims: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). Methods and Results: TMC0356 cultured in deMan–Rogosa–Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat‐killed TMC0356 were tested for their ability to induce interleukin (IL)‐12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N‐acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat‐killed TMC0356 significantly induced IL‐12 production in J774.1 cells and exhibited enhanced resistance to N‐acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL‐12 production in J774.1 cells were also associated. Conclusions: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL‐12 production in macrophages. Significance and Impact of the Study: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.     

Prevalence of arthropod antibodies in Korean patients with allergic rhinitis.

Arthropod antigens are main causative agents which induce allergic responses in humans. However, little information is known about the prevalence of specific arthropod allergens in Koreans with allergic diseases. The current study was designed to determine the positive rates of arthropod antibodies by the Korean inhalant panel of MAST-CLA. One hundred sixty patients, who were diagnosed with allergic rhinitis from an out-patient center at the Soonchunhyang University Chunan Hospital, were studied between August 1998 to July 2000. The overall positive rate, at least more than one specific antibody of arthropods such as Dermatophagoides farinae (Df), Dermatophagoides pteronyssinus (Dp), and cockroach mix (Cm), was 46.9%. Each positive rate of Df, Dp, and Cm was 45.0%, 43.1%, and 8.8%, respectively. A significant agreement among arthropod allergens was observed (Df and Dp: 95.6%, Kappa = 0.911, P < 0.001). Our data supported the fact that arthropods were the most common allergens in Korean patients with allergic rhinitis; however, the MAST-CLA should be modified to increase specificity of arthropod allergens.     

Nerve growth factor biosynthesis: Isolation and characterization of a guinea pig prostate kallikrein

Guinea pig prostate contains one major soluble esteropeptidase activity. The protein has been purified and characterized and found to be a glycoprotein comprised of a single polypeptide chain. The molecular weight of the deglycosylated protein is approximately 26,000. The esteropeptidase has a similar K_m for lysine and arginine synthetic substrates, although the V_max for arginine is much greater than that for lysine. Amino-terminal sequence analysis has also revealed a marked degree of homology to mouse γ-nerve growth factor (NGF) and the kallikrein family of serine proteases. In contrast to γ-NGF, however, the guinea pig enzyme does not appear to form stable complexes with β-NGF.     

Isolation and monolayer culture of guinea pig pancreatic duct epithelial cells

Summary Monolayers of cultured epithelial cells have been prepared from fragments of guinea pig pancreatic excretory ducts isolated by a simple procedure employing collagenase digestion and manual selection, through which virtually all of the ductal system can be recovered. The isolated fragments were cultured in enriched Waymouth's medium on extracellular matrices of various composition and thickness, including: thin (<5 μm) and thick (0.5 mm) layers of rat tail collagen; thin layers of human placental collagen; thin layers of Matrigel (a reconstituted basement membrane material); uncoated tissue culture plastic; and the cellulose ester membranes of Millipore Millicells. Cells spread rapidly from duct fragments cultured on uncoated plastic or on plastic coated with thin layers of rat tail collagen or human placental collagen and formed epithelial monolayers. However, these cells were squamous and lacked the abundant basolateral membrane amplification and apical microvilli characteristic of freshly isolated duct epithelial cells. Cells did not spread from duct fragments cultured on Matrigel. In contrast, when fragments of pancreatic ducts were explanted onto either a thick layer of rat tail collagen or onto Millicell membranes, cells readily spread and formed confluent monolayers of cuboidal epithelial cells characterized by abundant mitochondria, apical microvilli, and basolateral plasma membrane elaboration. These results demonstrate that different forms of extracellular matrix modulate the growth and differentiation of pancreatic duct epithelial cells, and that culture on a permeable substrate markedly enhances the maintenance of differentiated characteristics in this cell type. The monolayers formed on Millicell membranes should provide a useful model system for physiologic analysis of the regulation of electrolyte secretion by this epithelium. This research was supported by grants DK32994 and DK35912 from the National Institutes of Health, Bethesda, MD.     

Influence of degree of specific allergic sensitivity on severity of rhinitis and asthma in Chinese allergic patients

Background

The clinical manifestations of severe asthma are heterogeneous. Some individuals with severe asthma develop irreversible fixed airway obstruction, which is associated with poor outcomes. We therefore investigated the factors associated with fixed airway obstruction in Korean patients with severe asthma.

Methods

Severe asthma patients from a Korean adult asthma cohort were divided into two groups according to the results of serial pulmonary function tests. One group had fixed airway obstruction (FAO) [forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ratio < 0.7, n = 119] and the other had reversible airway obstruction (RAO) [FEV1/FVC ratio ≥ 0.7, n = 116]. Clinical and demographic parameters were compared between the two groups.

Results

Multivariate analysis showed that longer duration of disease, greater amount of cigarette smoking and absence of rhinosinusitis were significantly related to the development of FAO in severe asthmatics. Other parameters, including atopic status, pattern of airway inflammatory cells in induced sputum, and frequency of asthma exacerbations did not differ between the FAO and RAO groups.

Conclusion

Severe asthma patients with longer disease duration and the absence of rhinosinusitis are more likely to develop FAO. This study also demonstrates the importance of quitting smoking in order to prevent irreversible airway obstruction. Further investigation is required to determine the mechanism by which these factors can modify the disease course in Korean patients with severe asthma.     

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes

Summary We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, K_m values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on K_m and V_max indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID₅₀ values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.     

Fluoride or/and aluminum induced toxicity in guinea pig teeth with the low expression of dentine phosphoprotein

This study investigated the damage and expression of dentine phosphoprotein (DPP) in guinea pig teeth by the administration of fluoride (F) or/and aluminum (Al). Fifty‐two guinea pigs were divided randomly into four groups (control, F, Al, and F+Al). F (150 mg NaF/L) or/and Al (300 mg AlCl₃/L) were added in their drinking water for 90 days. The levels of F ion, dentine sialophosphoprotein (DSPP) gene, and DPP protein in incisor and molar were determined, respectively. The results showed that the concentrations of F ion in F and F+Al groups were increased significantly. F induced the mottled enamel and irregular abrasion of teeth, which might occur as a consequence of depressed DSPP mRNA and DPP protein expression. Both the gene and protein expressions showed obvious decrease induced by Al, especially by F. There were no synergistic effects between F and Al, instead, Al inhibited the toxicity of F.     

Effects of aging on cholinephosphotransferase activity on guinea pig lung mitochondria and microsomes

It is known that the composition of phospholipids in lung changes with age. The final step in thede novo synthesis of phosphatidylcholine, a major component of lung surfactant, by the CDP-choline pathway, requires the enzyme cholinephosphotransferase (CPT). Even though CPT has earlier been proposed to be located exclusively in the endoplasmic reticulum, we have recently demonstrated its presence also in the mitochondria. We have earlier reported a gestational variation of CPT activity in fetal mitochondria and microsomes. In the present study we examined the subcellular distribution of CPT activity in lung as a function of age. After birth, the microsomal CPT activity continued to increase until adulthood (24 wks of age), thereafter it gradually decreased. On the otherhand, the CPT activity of mitochondria continued to increase with the advancement of age and beyond 72 wks of age, it was approximately 2-fold higher than that of the microsomal fraction.     

Longitudinal study of effects of oral dosage of Bifidobacterium bifidum G9‐1 on Japanese cedar pollen‐induced allergic nasal symptoms in guinea pigs

Previous studies using experimental animal models have reported the beneficial effects of probiotics on allergic responses; however, their long‐term effects on allergic nasal symptoms in clinical settings have not yet been elucidated in detail. In the present study, a guinea pig allergic rhinitis model involving repeated inhalation challenges with a natural allergen, Japanese cedar pollen, was used to examine the longitudinal effects of Bifidobacterium bifidum G9‐1 (BBG9‐1) on allergic nasal symptoms. BBG9‐1 was administered orally once a day. Amelioration of nasal blockage was consistently observed throughout the experimental period in the BBG9‐1‐treated group. Although challenge‐induced sneezing was not significantly inhibited in the BBG9‐1‐treated group, prolonged treatment with BBG9‐1 slightly reduced the frequency of sneezing. Antigen‐specific IgE antibody production was also not inhibited in the BBG9‐1‐treated group. Increases in the numbers of eosinophils and neutrophils in nasal cavity lavage fluid collected after pollen challenge were almost completely suppressed by BBG9‐1 treatment, whereas those in mast cell mediators, histamine and cysteinyl leukotrienes were not. In contrast, increases in the levels of nitric oxide metabolites were potently suppressed. Furthermore, prolonged BBG9‐1 treatment markedly suppressed exogenous leukotriene D₄‐induced nasal blockage. Thus, prolonged oral administration of BBG9‐1 suppresses Japanese cedar pollen‐induced allergic nasal symptoms. The inhibitory mechanisms responsible may involve reductions in the responsiveness of target organs, such as endothelial cells in nasal mucosal blood vessels, to chemical mediators.     

Exhaled breath profiling by electronic nose enabled discrimination of allergic rhinitis and extrinsic asthma

Aim: To assess whether an e-nose could discriminate between subjects affected by allergic rhinitis with and without concomitant extrinsic asthma, as well as from healthy controls, in terms of exhaled VOC-profile.

Methods: Fourteen patients with Extrinsic Asthma and Allergic Rhinitis (AAR), 14 patients with Allergic Rhinitis without asthma (AR) and 14 healthy controls (HC) participated in a cross-sectional study. Exhaled breath was collected by a standardized method and sampled by an e-nose (Cyranose 320). Raw data were reduced by Principal component analysis and analyzed by canonical discriminant analysis. Cross-validation accuracy (CVA) and Receiver Operating Characteristic(ROC)-curves were calculated. External validation in newly recruited patients (7 AAR, 7?AR and 7 HC) was tested using the previous training model.

Results: Breathprints of patients with AR clustered from those with AAR (CVA?=?85.7%), as well as HC (CVA?=?82.1%). Breathprints from AAR were also separated from those of HC (CVA?=?75.0%). External validation confirmed the above findings.

Conclusions: An e-nose can discriminate exhaled breath from subjects with allergic rhinitis with and without extrinsic asthma, which represent two different diseases with partly overlapping features. This supports the view of using breath profiling to diagnose asthma also in patients with allergic rhinitis.     

Diminished levels of nasal S100A7 (psoriasin) in seasonal allergic rhinitis: an effect mediated by Th2 cytokines

Background

S100A7 is an antimicrobial peptide involved in several inflammatory diseases. The aim of the present study was to explore the expression and regulation of S100A7 in seasonal allergic rhinitis (SAR).

Methods

Nasal lavage (NAL) fluid was obtained from healthy controls before and after lipopolysaccharide (LPS) provocation, from SAR patients before and after allergen challenge, and from SAR patients having completed allergen-specific immunotherapy (ASIT). Nasal biopsies, nasal epithelial cells and blood were acquired from healthy donors. The airway epithelial cell line FaDu was used for in vitro experiments. Real-time RT-PCR and immunohistochemistry were used to determine S100A7 expression in nasal tissue and cells. Release of S100A7 in NAL and culture supernatants was measured by ELISA. The function of recombinant S100A7 was explored in epithelial cells, neutrophils and peripheral blood mononuclear cells (PBMC).

Results

Nasal administration of LPS induced S100A7 release in healthy non-allergic subjects. The level of S100A7 was lower in NAL from SAR patients than from healthy controls, and it was further reduced in the SAR group 6 h post allergen provocation. In contrast, ASIT patients displayed higher levels after completed treatment. S100A7 was expressed in the nasal epithelium and in glands, and it was secreted by cultured epithelial cells. Stimulation with IL-4 and histamine repressed the epithelial S100A7 release. Further, recombinant S100A7 induced activation of neutrophils and PBMC.

Conclusions

The present study shows an epithelial expression and excretion of S100A7 in the nose after microbial stimulation. The levels are diminished in rhinitis patients and in the presence of an allergic cytokine milieu, suggesting that the antimicrobial defense is compromised in patients with SAR.     

Cytochemical localization of membrane-bound Mg2+-dependent ATPase activity in guinea pig sperm head before and during the acrosome reaction

The distribution of ATPase activity in the heads of uncapacitated, capacitated, and acrosome-reacting guinea-pig spermatozoa was examined cytochemically using the Wachstein-Meisel's technique. In uncapacitated spermatozoa, the reaction products of the enzyme activity were localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity was Mg²⁺-dependent and inhibited by both Ca²⁺ and SH-blocking agents. This Mg²⁺-dependent ATPase activity was also demonstrated at the same sites in capacitated spermatozoa, whereas it was completely absent in acrosome-reacting spermatozoa. Although we did not determine the exact time of inactivation of the enzyme, it appeared to occur before the plasma membrane fused with the underlying outer acrosomal membrane. The abrupt loss of the Mg²⁺-dependent ATPase activity in the plasma and outer acrosomal membranes immediately before the onset of the acrosome reaction seems to suggest that inactivation of this enzyme by Ca²⁺ is one of the important biochemical events involved in the acrosome reaction.     

Dithiothreitol,a disulfide-reducing agent,inhibits capacitation,acrosome reaction,and interaction with eggs by guinea pig spermatozoa

Capacitation of guinea pig spermatozoa in vitro was inhibited by the disulfide-reducing agent dithiothreitol (DTT). Even a brief treatment with DTT inhibited capacitation unless an oxidizing agent (glutathione disulfide) was present in the posttreatment medium. Precapacitated spermatozoa were unable to undergo the acrosome reaction in the presence of DTT, indicating that this reagent also blocks the acrosome reaction. Acrosome-reacted spermatozoa were incapable of attaching to and penetrating the zona pellucida in the presence of DTT. Even when acrosome-reacted spermatozoa were directly brought to the surface of zona-free eggs, they were unable to bind to and fuse with the egg plasma membrane so long as DTT was present in the medium. These observations suggest that the tertiary and quaternary structures of sperm surface proteins regulated by their thioldisulfide status are of critical importance in the physiology and function of spermatozoa preliminary to and in the process of fertilization.     

On the role of glucose in capacitation and acrosomal reaction of guinea pig sperm

In order to determine whether metabolizable sugars delayed capacitation of guinea pig spermatozoa, these cells were pre-incubated in Tyrode's pyruvate lactate glucose medium (T-PLG) or Tyrode's glucose solution (T-G). They were then transferred to minimal culture medium containing pyruvate and lactate (MCM-PL) and the occurrence of acrosomal reactions (AR) was determined by light microscopic observations of wet mount aliquots. The percentage of acrosomal reactions was quantitated in fixed samples and occurrence of a true AR was confirmed by electron microscopy. Activated acrosome-reacted spermatozoa were observed within 5 min when cells were transferred to MCM-PL solution, after preincubating them for 60–120 min either in T-PLG or T-G media. By 15 min in MCM-PL the percentage of acrosome-reacted spermatozoa reached values similar to those obtained in cells pre-incubated from the beginning in MCM-PL medium (P > 0.05 in both) but significantly different from T-PLG and T-G controls (P < 0.0005 in both). The acrosomal reaction was external calcium dependent and independent of the Tyrode's media pH ranging from 7.2 to 8.0. The results obtained suggested that capacitation occurred in T-PLG and that it was not delayed by glucose; the results also suggested that capacitation could occur within a short time with glucose as the only exogenous substrate, but that the acrosome reaction could have been arrested by a glucose metabolite. Data are presented which suggest that intracellular levels of glucose-6-phosphate (as 2-deoxyglucose-6-phosphate)could play a key role in the expression of the acrosome reaction in sperm already able to perform it. A new hypothesis is suggested for the development of the fertilizing potential of guinea pig sperm when in the female genital tract.