Biomedical subjects

Hirohito Kita

Publications and source records attributed to Hirohito Kita.

At least 19 recordsLinked to original sources

PubMed2006

Allergen-specific in vitro cytokine production in adult patients with eosinophilic esophagitis.

Although the pathogenesis of eosinophilic esophagitis (EE) likely involves hypersensitivity reactions against exogenous allergens, allergen-specific cellular immune responses have not been studied. We investigated allergen-induced cytokine production by peripheral blood mononuclear cells (PBMCs) in adult patients with EE (n=15) and healthy controls (HC; n=9). PBMCs were incubated with nine common food and environmental allergens or a nonspecific mitogen, and the levels of interleukin (IL)-5, IL-10, IL-13, and interferon-gamma in the cell-free supernatants were determined. Spontaneous and mitogen-stimulated cytokine production did not differ between EE and HC. House dust mite, ragweed, Aspergillus, milk, and soy induced significantly higher IL-5 production in EE (P < 0.05). House dust mite also augmented IL-13 production in EE (P < 0.05). Furthermore, PBMCs from three EE patients without allergen-specific IgE vigorously produced IL-5 and IL-13 on allergen stimulation. Thus, immune responses in EE are characterized by enhanced production of Th2-like cytokines against both food and environmental allergens.

Adult↗

PubMed2006

CD38-deficient mice have reduced airway hyperresponsiveness following IL-13 challenge.

The transmembrane glycoprotein CD38 in airway smooth muscle is the source of cyclic-ADP ribose, an intracellular calcium-releasing molecule, and is subject to regulatory effects of cytokines such as interleukin (IL)-13, a cytokine implicated in asthma. We investigated the role of CD38 in airway hyperresponsiveness using a mouse model of IL-13-induced airway disease. Wild-type (WT) and CD38-deficient (CD38KO) mice were intranasally challenged with 5 microg of IL-13 three times on alternate days under isoflurane anesthesia. Lung resistance (R(L)) in response to inhaled methacholine was measured 24 h after the last challenge in pentobarbital-anesthetized, tracheostomized, and mechanically ventilated mice. Bronchoalveolar cytokines, bronchoalveolar and parenchymal inflammation, and smooth muscle contractility and relaxation using tracheal segments were also evaluated. Changes in methacholine-induced R(L) were significantly greater in the WT than in the CD38KO mice following intranasal IL-13 challenges. Airway reactivity after IL-13 exposure, as measured by the slope of the methacholine dose-response curve, was significantly higher in the WT than in the CD38KO mice. The rate of isometric force generation in tracheal segments (e.g., smooth muscle reactivity) was greater in the WT than in the CD38KO mice following incubation with IL-13. IL-13 treatment reduced isoproterenol-induced relaxations to similar magnitudes in tracheal segments obtained from WT and CD38KO mice. Both WT and CD38KO mice developed significant bronchoalveolar and parenchymal inflammation after IL-13 challenges compared with naïve controls. The results indicate that CD38 contributes to airway hyperresponsiveness in lungs exposed to IL-13 at least partly by increasing airway smooth muscle reactivity to contractile agonists.

ADP-ribosyl Cyclase 1↗

PubMed2006

Childhood asthma and measles vaccine response.

BACKGROUND: Asthmatic patients have a TH2-predominant milieu that is associated with humoral immunity. However, little is known about whether humoral immune responses to viral antigens differ between asthmatic and nonasthmatic children. OBJECTIVE: To determine whether humoral immune response differs in asthmatic patients vs nonasthmatic patients. METHODS: Measles virus specific IgG antibody levels were determined for the Rochester Family Measles Study cohort (n = 876), a convenience sample of healthy children 5 to 12 years of age in Rochester, MN. We conducted comprehensive medical record reviews of 838 children who were eligible for this study. We determined the child's asthma status at the time of determination of antibody levels by applying predetermined criteria for asthma. Comparisons were made using the 2-sample t test or chi2 test. RESULTS: Of the 838 children, 156 (18.6%) had asthma at the time of the determination of antibody levels and were not taking systemic steroids within 14 days of specimen collection. Among those with a nonequivocal antibody reading, the seropositive response rates were similar in asthmatic patients and nonasthmatic patients (89.7% vs 90.3%, respectively; P = .83). However, the equivocal response rates were slightly higher among asthmatic patients than nonasthmatic patients (6.4% vs 4.7%, respectively). CONCLUSION: Asthmatic children seem to have similar humoral immune responses to measles vaccine as those without asthma. Although the findings reassure health care practitioners, whether this finding is generalizable to other vaccines and whether asthmatic patients with low antibody levels have normal cell-mediated immunity need to be elucidated in future studies.

Antibody Formation↗

PubMed2006

Intranasal exposure to staphylococcal enterotoxin B elicits an acute systemic inflammatory response.

Staphylococcus aureus produces a variety of superantigen exotoxins, including staphylococcal enterotoxin B (SEB). Little is known regarding the pathogenesis of SEB entering through the intranasal route. Intranasal exposure to SEB might occur because of nasal packing following surgical procedure, biologic warfare, or even S. aureus colonization. We evaluated the local and systemic effects of intranasally delivered SEB using a series of human leukocyte antigen (HLA) class II transgenic mice as conventional mice expressing endogenous class II molecules mount a poor immune response to SEB. Gene expression profiling using microarrays showed robust up-regulation of genes involved in several proinflammatory pathways as early as 3 h post-intranasal challenge with SEB in HLA class II transgenic mice. This was accompanied by a several hundred-fold increase in serum levels of pro-inflammatory cytokines such as IL-12, IL-6, TNF-alpha, IFN-gamma, as well as MCP-1 in HLA class II transgenic mice but not in C57BL/6 mice; CD4 or CD8 T-cells independently contributed to the systemic cytokine response. Defective IL-12 or IL-4 receptor signaling significantly decreased or increased serum IFN-gamma, respectively. Intranasal exposure to SEB resulted in neutrophil influx into bronchoalveolar lavage fluid and caused expansion of both CD4 and CD8 T-cells expressing TCR V beta 8 in the spleen. This was accompanied by mononuclear cell infiltration in the liver reminiscent of the systemic inflammatory response syndrome. Thus, we have shown, for the first time, that intranasal administration of SEB can cause systemic immune activation.

Administration, Intranasal↗

PubMed2006

Intranasal exposure to bacterial superantigens induces airway inflammation in HLA class II transgenic mice.

Staphylococcus aureus is widely prevalent in the nasopharynges of healthy individuals (carriers) but can also cause serious infections. S. aureus can elaborate a variety of superantigen exotoxins in "carrier" or "pathogenic" states. Streptococcus pyogenes can also colonize the nasopharynx and elaborate superantigens. Unlike the acute effects of superantigen exotoxins absorbed through the gut or vaginal mucosa, little is known regarding the pathogenesis of superantigens entering through the intranasal route. In the current study, we evaluated the local and systemic effects of staphylococcal enterotoxin B (SEB) and streptococcal pyrogenic exotoxin A (SPEA) delivered through the intranasal route. Superantigens were administered intranasally on multiple occasions, and experimental animals were sacrificed on day 8 for experimental analyses. SEB-induced airway inflammation was more pronounced for HLA-DR3 transgenic mice than for BALB/c mice, consistent with bacterial superantigens binding more efficiently to human than murine major histocompatibility complex class II. The nature of the airway inflammation in HLA-DR3 mice was determined by the concentration of SEB applied intranasally. Low concentrations (20 ng) induced eosinophilic airway inflammation as well as eosinophil degranulation, whereas intranasal exposure to higher concentrations (2,000 ng) resulted in neutrophilic airway inflammation, permanent airway destruction, toxic shock, and mortality. SEB-induced eosinophilic inflammatory response was enhanced in signal transducer and activator of transcription (STAT)-4-deficient HLA-DQ8 transgenic mice with defective interleukin-12 signaling. Intranasal administration of SPEA induced airway inflammation and systemic immune activation in HLA-DQ8 transgenic mice. In conclusion, repeated chronic intranasal exposure to bacterial superantigens causes airway inflammation and systemic immune activation.

Administration, Intranasal↗

PubMed2006

The role of ubiquitous airborne fungi in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a confusing disease for both allergists and otorhinolaryngologists, partly because of its poorly understood pathophysiology and partly because of its limited treatment options. Several recent reports have provided evidence for a better understanding of the etiology and the relationship of CRS to airborne fungi-especially to Alternaria. First, the development of novel methods enables detection of certain fungi in mucus from the nasal and paranasal sinus cavities. Second, a non-IgE-mediated immunological mechanism for reactivity of patients with CRS to certain common fungi has been described. Third, these fungi are surrounded by eosinophils in vivo, suggesting that they are targeted by eosinophils. Finally, the preliminary results of studies using antifungal agents to treat patients with CRS are promising. Overall, these recent discoveries provide a logical mechanism for the pathophysiology of CRS, and they also suggest promising avenues for treatment of CRS with antifungal agents.

Air Microbiology↗

PubMed2005

Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils.

Eosinophils and their products are probably important in the pathophysiology of allergic diseases, such as bronchial asthma, and in host immunity to certain organisms. An association between environmental fungal exposure and asthma has been long recognized clinically. Although products of microorganisms (e.g., lipopolysaccharides) directly activate certain inflammatory cells (e.g., macrophages), the mechanism(s) that triggers eosinophil degranulation is unknown. In this study we investigated whether human eosinophils have an innate immune response to certain fungal organisms. We incubated human eosinophils with extracts from seven environmental airborne fungi (Alternaria alternata, Aspergillus versicolor, Bipolaris sorokiniana, Candida albicans, Cladosporium herbarum, Curvularia spicifera, and Penicillium notatum). Alternaria and Penicillium induced calcium-dependent exocytosis (e.g., eosinophil-derived neurotoxin release) in eosinophils from normal individuals. Alternaria also strongly induced other activation events in eosinophils, including increases in intracellular calcium concentration, cell surface expression of CD63 and CD11b, and production of IL-8. Other fungi did not induce eosinophil degranulation, and Alternaria did not induce neutrophil activation, suggesting specificity for fungal species and cell type. The Alternaria-induced eosinophil degranulation was pertussis toxin sensitive and desensitized by preincubating cells with G protein-coupled receptor agonists, platelet-activating factor, or FMLP. The eosinophil-stimulating activity in Alternaria extract was highly heat labile and had an M(r) of approximately 60 kDa. Thus, eosinophils, but not neutrophils, possess G protein-dependent cellular activation machinery that directly responds to an Alternaria protein product(s). This innate response by eosinophils to certain environmental fungi may be important in host defense and in the exacerbation of inflammation in asthma and allergic diseases.

Alternaria↗

PubMed2005

Effects of the very late adhesion molecule 4 antagonist WAY103 on human peripheral blood eosinophil vascular cell adhesion molecule 1-dependent functions.

BACKGROUND: Eosinophil infiltration to the lung in allergic inflammation can be initiated by the tethering of circulating cells through very late adhesion molecule 4 (VLA-4; alpha4beta1, CD49d/CD29) to vascular cell adhesion molecule 1 (VCAM-1) expressed on pulmonary vascular endothelium. Small-molecule VLA-4 antagonists have been proposed as a therapeutic mechanism to prevent eosinophil infiltration in asthma; however, they might affect other eosinophil functions. OBJECTIVE: The small-molecule VLA-4 antagonist (2S)-3-(4-Dimethylcarbamoyloxyphenyl)-2-{[(4R)-5,5-dimethyl-3-(1-methyl-1H-pyrazole-4 sulfonyl)thiazolidine-4-carbonyl]amino}propionic acid (WAY103) was assessed for its effects on eosinophil VLA-4-dependent functions, including adhesion, migration, respiratory burst, and degranulation. METHODS: Human peripheral blood eosinophils were preincubated with WAY103, anti-alpha4, and/or anti-beta2 integrin mAbs and then assessed for adhesion to recombinant VCAM-1, intercellular adhesion molecule 1, and endothelial cell monolayers. Transmigration was measured by using human pulmonary microvascular endothelial cell monolayers and Transwell filters. Superoxide anion generation was determined by means of cytochrome C reduction and degranulation by means of eosinophil-derived neurotoxin release. RESULTS: WAY103 inhibition of eosinophil adhesion to recombinant VCAM-1 was dose dependent (63% inhibition with 100 nM WAY103, P < .04) and comparable with inhibition caused by anti-alpha4 mAb (60.1% inhibition). Although pretreatment with WAY103 also decreased eosinophil adhesion to TNF-alpha plus IL-4-activated human pulmonary microvascular endothelial cell monolayers, it did not prevent eosinophil transendothelial migration in response to RANTES. Finally, WAY103 inhibited VCAM-1-stimulated superoxide generation but enhanced cytokine-activated eosinophil-derived neurotoxin degranulation. CONCLUSION: Although small-molecule VLA-4 antagonists, such as WAY103, might reduce eosinophil adhesion, this approach might not be sufficient to eliminate this cell from in vivo allergic airway inflammatory participation and could even promote specific cell activation.

Adolescent↗

PubMed2005

The synthetic chemoattractant peptide WKYMVm induces superoxide production by human eosinophils via the phosphoinositide 3-kinase-mediated activation of ERK1/2.

BACKGROUND: Eosinophils play a key role in allergic inflammation and parasitic infections. The synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), has been previously shown to activate eosinophils and thus to enhance respiratory burst through the formyl peptide receptors. OBJECTIVE: This study was undertaken to determine the intracellular signaling pathway involved in WKYMVm-stimulated superoxide production by human eosinophils. METHODS: Purified eosinophils from peripheral blood were stimulated with various concentrations (10(-3) to 10 microM) of WKYMVm and the involvement of PI3-kinase and MAP kinases in WKYMVm-triggered superoxide production was investigated using pharmacological inhibitors. RESULTS: WKYMVm-induced superoxide production by eosinophils was strongly inhibited by pretreatment with the PI3-kinase inhibitor LY294002. In addition, pretreatment with the ERK1/2 kinase inhibitor PD98059 resulted in marked inhibition of superoxide production induced by WKYMVm. Indeed, WKYMVm strongly induced phosphorylation of ERK1/2. The ERK1/2 activation by the peptide was transient and peaked after 2 min of stimulation. Furthermore, ERK1/2 activation by WKYMVm was completely inhibited by pretreatment with the PI3-kinase inhibitor LY294002, but not by the PKC inhibitor Ro-31-8220. CONCLUSION: These results suggest that WKYMVm stimulates human eosinophils to induce superoxide production via a PI3-kinase-mediated ERK1/2 pathway.

Blotting, Western↗

PubMed2005

The role of ubiquitous airborne fungi in chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) is a confusing disease for both allergists and otorhinolaryngologists, partially due to its poorly understood pathophysiology and partially due to its limited treatment options. Several recent reports now provide evidence for a better understanding of the etiology and the relationship of CRS to airborne fungi, especially to Alternaria. First, the development of novel methods enables detection of certain fungi in mucus from the nasal and paranasal sinus cavities. Second, a non-immunoglobulin E-mediated immunologic mechanism for reactivity of CRS patients to certain common fungi has been described. Third, these fungi are surrounded by eosinophils in vivo, suggesting that they are targeted by eosinophils. Fourth, the preliminary results of studies using antifungal agents to treat patients with CRS are promising. Overall, these recent discoveries provide a logical mechanism for the pathophysiology of CRS, and they also suggest promising avenues for treatment of CRS with antifungal agents.

Air Microbiology↗

PubMed2005

Treatment of chronic rhinosinusitis with intranasal amphotericin B: a randomized, placebo-controlled, double-blind pilot trial.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common chronic diseases. Its etiology is unknown, and there is a paucity of effective medical treatments. OBJECTIVE: We tested the hypothesis that intranasal antifungal treatment improves the objective computed tomography (CT) findings (inflammatory mucosal thickening), nasal endoscopy stages, and symptoms of CRS. METHODS: A randomized, placebo-controlled, double-blind, single-center trial used amphotericin B to treat 30 randomly selected patients with CRS. Patients were instructed to instill 20 mL amphotericin B (250 mug/mL) or placebo to each nostril twice daily for 6 months. The primary outcome was a quantitative reduction in inflammatory mucosal thickening on CT scans of a standardized coronal cut. Secondary outcome measures were endoscopic scores, patient symptom scores, and levels of intranasal inflammatory mediators. RESULTS: Twenty-four patients completed the 6 months of treatment. Patients receiving amphotericin B achieved a relative reduction in the percentage of mucosal thickening on CT scans (n = 10; -8.8%) compared with placebo (n = 14; +2.5%; P = .030). Likewise, the changes in the endoscopic scores improved in the amphotericin B group compared with placebo ( P = .038). Between-group comparisons of the changes in the intranasal mucus levels of eosinophil-derived neurotoxin showed a reduction in the amphotericin B group and an increase in the placebo group ( P = .046); levels of IL-5 showed similar tendencies ( P = .082). CONCLUSION: Intranasal amphotericin B reduced inflammatory mucosal thickening on both CT scan and nasal endoscopy and decreased the levels of intranasal markers for eosinophilic inflammation in patients with CRS.

Administration, Intranasal↗

PubMed2005

Striking deposition of toxic eosinophil major basic protein in mucus: implications for chronic rhinosinusitis.

BACKGROUND: The mechanisms by which eosinophilic inflammation damages the epithelium and contributes to recurrent acute exacerbations in chronic rhinosinusitis (CRS) have not been fully elucidated. OBJECTIVE: We tested the hypotheses that eosinophils deposit toxic major basic protein (MBP) in the mucus and that MBP reaches concentrations able to damage the sinonasal epithelium. METHODS: Tissue specimens with mucus attached to the tissue were carefully collected from 22 patients with CRS and examined by using immunofluorescence staining for MBP. This immunofluorescence was digitally analyzed to determine the area covered by MBP and the intensity of the staining (estimating MBP concentration). Levels of MBP in extracts from nasal mucus were quantitated by means of RIA. RESULTS: Heterogeneous eosinophilia was evident within tissue and mucus specimens. All tissue specimens showed intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens showed diffuse MBP throughout and abundant diffuse extracellular MBP deposition within clusters of eosinophils. Digitized analyses of MBP immunofluorescence revealed increased area coverage (P < .0001) in mucus compared with that seen in tissue. Estimated concentrations of MBP within the clusters suggested toxic levels. MBP concentrations in mucus extract reached 11.7 microg/mL; MBP was not detectable in healthy control subjects. CONCLUSION: In patients with CRS, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epithelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium from the luminal side and could predispose patients with CRS to secondary bacterial infections.

Adolescent↗

PubMed2005

Dendritic cells activated by cross-linking B7-DC (PD-L2) block inflammatory airway disease.

BACKGROUND: Allergic asthma is an increasing clinical problem that might be addressed with immunologically based interventions. A novel human antibody that activates human and mouse dendritic cells (DCs) by cross-linking B7-DC has strong immunomodulatory properties and blocks antigen-induced inflammatory lung disease in mice. OBJECTIVE: We sought to evaluate the ability of activated DCs to mediate an antibody-induced protective response against inflammatory lung disease. METHODS: An adoptive transfer strategy was used to test the ability of antibody treatments to activate DCs, inducing a protective response against inflammatory lung disease in mice presensitized to ovalbumin (OVA). After transfer of activated DCs, recipient mice were exposed repeatedly to airway antigen and evaluated for changes in immune reactivity and airway inflammatory disease. RESULTS: Animals presensitized to OVA receiving either systemic treatments with B7-DC cross-linking antibody (XAb) or adoptively transferred antibody-activated DCs were completely protected from airway inflammatory responses normally induced by repeated exposure to OVA. Lymphocytes isolated from spleens or lung-draining lymph nodes of treated animals were highly responsive to OVA and secreted TNF-alpha, IFN-gamma, and IL-10. In contrast, IL-4 was not produced by cells isolated from animals receiving B7-DC XAb. CONCLUSION: Activation of DCs with B7-DC XAb ex vivo before adoptive transfer into presensitized mice is sufficient to protect animals completely from inflammatory lung disease induced by subsequent repeated airway exposure to the offending antigen. This finding is consistent with our hypothesis that in vivo administration of B7-DC XAb modulates the immune response by activating endogenous DCs.

Adoptive Transfer↗

PubMed2005

Secretory IgA induces antigen-independent eosinophil survival and cytokine production without inducing effector functions.

BACKGROUND: Eosinophils in human beings reside in tissues, especially the mucosal tissues of the gastrointestinal tract and inflamed airways. Secretory IgA (S-IgA) is the predominant antibody secreted by these tissues and likely plays a role in the innate immune response. OBJECTIVE: Because eosinophils and S-IgA are often colocalized in mucosal tissues, we examined the potential regulatory effects of S-IgA without antigens on survival, gene expression, and effector functions of human eosinophils. METHODS: Eosinophils were incubated with S-IgA in solution without antigens (soluble S-IgA) or with S-IgA immobilized to mimic multivalent antigen cross-linking. Eosinophil activation was monitored by superoxide anion generation and degranulation. Survival was assessed between 24 and 96 hours. Gene and protein expression were examined by microarray and ELISA. Eosinophil lysates were examined by immunoblot for extracellular signal-regulated kinase (ERK) phosphorylation. RESULTS: Immobilized S-IgA stimulated eosinophil superoxide production and degranulation; soluble S-IgA did not. Although immobilized S-IgA inhibited eosinophil survival in vitro, soluble S-IgA enhanced survival; this involved autocrine production of GM-CSF. Soluble S-IgA without antigens induced increases in mRNA levels of various cytokines, chemokines, signal transduction molecules, antiapoptotic factors, and cell surface markers. By using ELISA, we confirmed protein expression of selected mediators. Eosinophil interaction with soluble S-IgA likely involves FcalphaRI (CD89) and ERK pathway activation. CONCLUSION: Secretory IgA without multivalent antigens may regulate survival and gene expression of eosinophils. Eosinophils in mucosal tissues can be either primed for action (cytokine production and survival) or fully activated (degranulation and superoxide release) by different forms of S-IgA.

Cell Degranulation↗

PubMed2005

Degranulation of human eosinophils induced by Paragonimus westermani-secreted protease.

Eosinophil degranulation is considered to be a key effector function for the killing of helminthic worms and tissue inflammation at worm-infected lesion sites. However, relatively little data are available with regard to eosinophil response after stimulation with worm-secreted products which contain a large quantity of cysteine proteases. In this study, we attempted to determine whether the degranulation of human eosinophils could be induced by the direct stimulation of the excretory-secretory products (ESP) of Paragonimus westermani, which causes pulmonary paragonimiasis in human beings. Incubation of eosinophils for 3 hr with Paragonimus-secreted products resulted in marked degranulation, as evidenced by the release of eosinophil-derived neurotoxin (EDN) in the culture supernatants. Moreover, superoxide anion was produced by eosinophils after stimulation of the ESP. The ESP-induced EDN release was found to be significantly inhibited when the ESP was pretreated with protease inhibitor cocktail or the cysteine protease inhibitor, E-64. These findings suggest that human eosinophils become degranulated in response to P. westermani-secreted proteases, which may contribute to in vivo tissue inflammation around the worms.

Animals↗

PubMed2004

Platelet-activating factor stimulates cytoplasmic alkalinization and granule acidification in human eosinophils.

The effects of platelet-activating factor (PAF) and IL-5 on intracellular pH were investigated in human eosinophils. Purified peripheral blood eosinophils were loaded with the ratiometric fluorescent pH indicator BCECF-AM ester. Stimulation of eosinophils with PAF produced time-dependent alkalinization of the cytoplasm from an initial pH of 7.1+/-0.04 to 7.5+/-0.05. A similar alkalinization response was produced by the calcium ionophore, ionomycin and by the calcium ATPase inhibitor, thapsigargin. These compounds as well as PAF produce significant increases in cytoplasmic calcium ([Ca2+]i). In contrast, IL-5 and the protein kinase C (PKC) activator phorbol myristate acetate (PMA) did not produce cytoplasmic alkalinization and had no effect on [Ca2+]i in eosinophils. PAF-stimulated alkalinization was not inhibited under conditions that blocked plasma membrane Na+-H+ exchange, proton channel or plasma membrane H+-ATPase activities. Measurements of intragranule pH with a cell permeant pH indicator (LysoSensor Yellow/Blue DND-160), which partitions into intracellular acidic compartments, revealed that PAF-stimulated cytosolic alkalinization correlated with intragranule acidification. These results suggest that the increase in [Ca2+]i after PAF stimulation activates a H+-ATPase present in the granule membranes, leading to enhanced granule acidification and cytoplasmic alkalinization. We propose that granule acidification is an important step in solubilization of major basic protein crystals, which are stored within the granule core, in preparation for degranulation and release of these proteins.

Adenosine Triphosphatases↗

PubMed2004

Blockade of allergic airway inflammation following systemic treatment with a B7-dendritic cell (PD-L2) cross-linking human antibody.

We present a novel immunotherapeutic strategy using a human B7-DC cross-linking Ab that prevents lung inflammation, airway obstruction, and hyperreactivity to allergen in a mouse model of allergic inflammatory airway disease. Dendritic cells (DC) have the ability to skew the immune response toward a Th1 or Th2 polarity. The sHIgM12 Ab functions in vitro by cross-linking the costimulatory family molecule B7-DC (PD-L2) on DC up-regulating IL-12 production, homing to lymph nodes, and T cell-activating potential of these APCs. Using chicken OVA as a model Ag, the administration of sHIgM12 Ab to BALB/c mice blocked lung inflammation, airway pathology, and responsiveness to methacholine, even after animals were presensitized and a Th2-polarized immune response was established. This therapeutic strategy was ineffective in STAT4-deficient animals, indicating that IL-12 production is critical in this system. Moreover, the polarity of the immune response upon in vitro restimulation with Ag is changed in wild-type mice, with a resulting decrease in Th2 cytokines IL-4 and IL-5 and an increase in the immunoregulatory cytokine IL-10. These studies demonstrate that the immune response of hypersensitized responders can be modulated using B7-DC cross-linking Abs, preventing allergic airway disease upon re-exposure to allergen.

Adjuvants, Immunologic↗

PubMed2004

Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester.

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP-PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a approximately 470 kDa PAPP-A form (PAPP-A470) to a approximately 400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.

Adult↗