High-dose cutaneous exposure to mite allergen induces IgG-mediated protection against anaphylaxis.

BACKGROUND: The relationship among natural allergen exposure, induction of blocking antibody and the occurrence of atopic allergy-particularly in the presence of IgE production-is debatable. OBJECTIVE: To clarify the relationship between the dose of cutaneous exposure to dust mite allergen and susceptibility to the IgE-mediated allergic response in relation to IgG production. METHODS: NC/Nga mice were epicutaneously exposed to various doses of Dermatophagoides pteronyssinus allergen to induce atopic dermatitis-like skin lesions. We then evaluated the skin lesions, induction of mite-specific immune responses, and susceptibility to anaphylaxis. RESULTS: Dose-dependent exacerbation of atopic dermatitis-like skin lesions and increases in mite-specific IgG and IgE production were observed. However, mice exposed to relatively low doses of mite allergen showed hypersusceptibility to mite allergen-specific anaphylaxis. We also showed that adoptive transfer of total IgG from Dp-sensitized mice rescued mice from the hypersusceptibility seen in those exposed to low doses of mite allergen. CONCLUSIONS AND CLINICAL RELEVANCE: High-dose cutaneous exposure to dust mites induced effective blocking IgG production, even if accompanied by IgE production. Our data might support the concept that an increase in IgG titre, not a decrease in IgE titre, is a marker of clinical improvement in allergen-specific immunotherapy.

A toll-like receptor 3 single nucleotide polymorphism in Japanese patients with sarcoidosis.

Toll-like receptor 3 (TLR3) may be associated with T helper 1 immune response. This study aimed to investigate the role of a functional TLR3 single nucleotide polymorphism (SNP) in sarcoidosis. We genotyped 220 Japanese patients with sarcoidosis and 140 controls for TLR3 SNP rs3775291 to analyze its association with susceptibility to sarcoidosis and assessed its relationship to clinical features in 172 patients over 2 years. The TLR3 rs3775291 genotype was not significantly associated with disease susceptibility. However, patients with cardiac sarcoidosis (CS) significantly more frequently had the TT genotype (p < 0.01) or the T allele (p < 0.05) than those patients without CS. We conclude that TLR3 SNP rs3775291 may affect cardiac involvement in Japanese patients with sarcoidosis.

Endpoints for clinical trials of sarcoidosis.

Over the past few years an increasing number of prospective controlled sarcoidosis treatment trials have been completed. Unfortunately, these studies utilize different endpoints making comparisons between studies difficult. At the recent World Association of Sarcoidosis and other Granulomatous disease (WASOG) meeting, a session was dedicated to the evaluation of clinical endpoints for various disease manifestations. These included pulmonary, pulmonary hypertension, fatigue, cutaneous, and a classification of clinical disease phenotypes. Based on the available literature and our current understanding of the disease, recommendations for clinical evaluation were proposed for each disease category. For example, it was recommended that pulmonary studies should include changes in the forced vital capacity. Additionally, it was recommended that all trials should incorporate measurement of quality of life.

A CD40 single-nucleotide polymorphism affects the lymphocyte profiles in the bronchoalveolar lavage of Japanese patients with sarcoidosis.

CD40 plays a critical role in adaptive immunity, and alveolar macrophages in patients with sarcoidosis express higher levels of CD40. This study investigated the association of rs1883832, a functional single-nucleotide polymorphism in the CD40 gene with susceptibility to sarcoidosis and phenotypes of sarcoidosis. Genotyping of rs1883832 in 175 Japanese patients with sarcoidosis and 150 age- and sex-matched controls revealed no significant difference between the genotypes of the patient and control groups (CC/CT/TT, 32.8/52.0/14.7% in the patients; 37.3/48.0/14.7% in the controls, P = 0.66; allele C, 59.1% in the patients, 61.3% in the controls, P = 0.57). T-cell and CD4+ cell counts in the bronchoalveolar lavage fluid were significantly higher in the TT genotype group than in the CC and CT genotype group.

Long-term changes in airway-wall thickness on computed tomography in asthmatic patients.

BACKGROUND: Effects of long-term treatment with inhaled corticosteroids (ICSs) on airway-wall thickness in patients with asthma remain unknown. OBJECTIVES: To determine whether airway-wall thickness consistently decreases after long-term ICS treatment, and to analyze factors contributing to long-term airway-wall changes in asthmatics. METHODS: A retrospective analysis of long-term changes in airway-wall thickness using computed tomography was performed in 14 patients with asthma. Wall area corrected by body surface area (WA/BSA) was examined at baseline, 12 weeks after the commencement of ICSs (second measurement), and at least 2 years (mean +/- SEM. 4.2 +/- 0.5) after the second measurement (third measurement). Mean +/- SEM changes in WA/BSA from the second to the third measurements were analyzed. RESULTS: The mean change in WA/BSA was not significant between the second and the third measurements (-0.27 +/- 0.59 mm2/m2/y). Overall, the changes were significantly associated with disease duration but not with other clinical indices. When the 14 patients were divided into 2 groups using a cutoff value of 0.32 mm2/m2/y for the mean change in WA/BSA, for the 5 patients whose WA/BSA exceeded this cutoff, daily ICS doses were not reduced and both forced expiratory volume in the first second (FEV1) and forced vital capacity decreased significantly. For the remaining 9 patients, daily ICS doses were reduced and long-term FEV1 values did not change. CONCLUSIONS: Despite long-term treatment with ICSs, airway-wall thickness did not consistently decrease. One possible mechanism underlying poor response to long-term treatment may be long-standing asthma.

Significance of plasma NT-proBNP levels as a biomarker in the assessment of cardiac involvement and pulmonary hypertension in patients with sarcoidosis.

BACKGROUND: Cardiac involvement and pulmonary hypertension (PH) are life-threatening complications in sarcoidosis. OBJECTIVE: This study aimed to investigate the utility of plasma NT-proBNP in the assessment of these conditions in sarcoidosis patients. STUDY DESIGN AND METHODS: A prospective, observational study was performed on 150 consecutive Japanese sarcoidosis patients. Doppler echocardiography was performed in all subjects, and those who were successfully evaluated for PH status were included in the analysis. Cardiac sarcoidosis was diagnosed based on Japanese guidelines, and PH was defined as estimated systolic pulmonary artery pressure (sPAP) > or = 35 mmHg. The diagnostic accuracy of NT-proBNP according to the presence of cardiac sarcoidosis and PH was assessed based on receiver-operator characteristic (ROC) curves. RESULTS: 130 subjects were successfully evaluated for PH status. Of these, 29 met the diagnostic criteria of cardiac sarcoidosis, and 21 were diagnosed with PH. Plasma NT-proBNP levels were significantly higher in patients with cardiac sarcoidosis (p < 0.0001). Stepwise regression analysis showed that presence of cardiac sarcoidosis, decreased ejection fraction and increased sPAP were all independently associated with higher plasma NT-proBNP levels. Plasma NT-proBNP showed good accuracy in identifying patients with cardiac sarcoidosis (area under the ROC curve; AURC = 0.913). However, even when patients with cardiac sarcoidosis were excluded, plasma NT-proBNP levels could not be used reliably to identify patients with PH (AURC = 0.681). CONCLUSION: In patients with sarcoidosis, plasma NT-proBNP levels are a useful biomarker to identify cardiac involvement, but not to identify PH.

CD24 gene exon 2 dimorphism does not affect disease susceptibility in Japanese sarcoidosis patients.

BACKGROUND: CD24 proteins are expressed on several inflammatory cells, and play an important role for the T-cell activation. OBJECTIVES: The aim of this study is to investigate the relationship of a CD24 gene polymorphism to disease susceptibility or clinical findings including bronchoalveolar lavage (BAL) cell profiles in Japanese sarcoidosis patients. METHODS: A previously reported functional single nucleotide polymorphism (SNP) of CD24 gene exon 2 was examined in 186 Japanese sarcoidosis patients and 146 sex and age-matched healthy controls using restriction fragment length polymorphism method. The distribution of genotypes was compared between the two groups. The association between genotypes or alleles and clinical features or BAL cell profiles was also examined. RESULTS: There were no significant differences in the distribution of genotypes or allele frequencies between sarcoidosis and controls. There were also no significant differences in clinical features or BAL cell profiles among patients with different genotypes of CD24. CONCLUSIONS: There was no relationship between a CD24 exon 2 SNP and disease susceptibility or clinical findings in Japanese sarcoidosis patients.

Long-term complications and prognosis of chronic beryllium disease.

BACKGROUND: Chronic beryllium disease (CBD) is a rare disease, and there are no previous reports that have followed CBD patients over several decades. Thus, the long-term complications and prognosis of this illness still remain unclear. OBJECTIVE: The aim of this study was to investigate long-term complications and prognosis of CBD patients. STUDY DESIGN AND METHODS: This was a retrospective study based on the medical records of all CBD patients diagnosed at Kyoto University Hospital between the period 1973 to the present day. Ultimately, ten patients whose diagnoses had been made during the period 1973 to 1977 were included. Long-term physiological and radiological change, complications and prognosis of these patients were investigated. RESULTS: Three patients completely remitted, and one died of cor-pulmonale. Among the remaining six patients, four have been followed up for more than thirty years in our institute. The majority developed mixed patterns of lung function impairment, cavity lesions of the lung, pneumothorax, and respiratory infections. CONCLUSIONS: Long-term prognosis of CBD was poor with several complications due to chronic parenchymal and airway lesions.

Differential activity to shapes under shape-from-motion condition in macaque middle temporal area.

To investigate the neural mechanisms of motion-defined shape processing, we recorded single unit activity in the middle temporal area (MT) while monkeys performed a shape discrimination task under the shape-from-motion (SFM) condition, where a motion cue is critical for shape perception. About 40% of MT neurons responded differentially to shapes under the SFM condition. The differential responses to shapes could not be explained by either the heterogeneous structure of the receptive field or the amount of motion signal. On the other hand, under the shape-from-luminance (SFL) condition, where a luminance cue is critical for shape perception, the proportion of neurons showing differential responses to shapes was smaller than that under the SFM condition and the magnitudes of differential responses themselves were weaker. Thus, the requirement for motion processing for shape perception may facilitate a differential response to shapes under the SFM condition. We compared neuronal activities during the shape discrimination task with those during the direction discrimination task under the SFM condition. Differential responses to shapes were observed more frequently during the shape discrimination task than during the direction discrimination task. Thus, the motion-defined shape processing influenced MT activity.

Regulation of phospholipase D activity by neutral lipids in egg-yolk phosphatidylcholine small unilamellar vesicles and by calcium ion in aqueous medium.

Hydrolysis activity of phospholipase D from Streptomyces chromofuscus (PLD) was studied in small unilamellar vesicles (SUV) of egg yolk phosphatidylcholine (PC). The enzyme was associated with PC-SUV in a Ca(2+)-dependent manner. Both apparent maximum velocity, Vmax(app), and reciprocal of apparent Michaelis constant, i.e., apparent binding constant, 1/Km(app), increased with Ca2+ concentration, and the maximum values of these kinetic parameters were obtained at about 20 microM Ca2+. Incorporation of 1,2-diacylglycerol (DAG), cholesterol (Chol) or alpha-tocopherol (Toc) into PC-SUV induced shift of the antisymmetric PO2- stretching band of PC to lower frequency. The neutral lipids in SUV brought about increase of the Vmax(app) value (Yamamoto et al. (1993) Biochim. Biophys. Acta 1145, 293-297). On the basis of these findings we discussed the regulation of PLD activity in terms of the Ca(2+)-dependent complex formation of PLD with SUV, and the enhancement of susceptibility of the P-O bond in PC molecule by neutral lipids.

Effects of 1,2-diacylglycerol and cholesterol on the hydrolysis activity of phospholipase D in egg-yolk phosphatidylcholine bilayers.

Effects of cholesterol (Chol) and 1,2-diacylglycerol (DAG) on the hydrolysis activity of phospholipase D (from Streptomyces chromofuscus) were studied in small unilamellar vesicles (SUV) of egg-yolk phosphatidylcholine (PC). 1,2-Diacylglycerol used here is derived from PC. Choline produced in the reaction was monitored by using a choline oxidase-oxygen electrode. Addition of 18.3 mol% Chol into SUV (2 mM PC) led to a small increase in the reaction rate. On the other hand, 18.3 mol% DAG in SUV brought about a 5-6-fold rate of choline production. The apparent maximum velocity, Vmax(app), increased by addition of DAG and Chol in SUV. In PC/Chol-SUV, the effect of increase in Vmax(app) was largely compensated by the increase in the apparent Michaelis constant, Km (app). The Chol and DAG molecules did not have significant effects on the kinetic parameters, when PC was solubilized in the micelles of heptaethylene glycol dodecyl ether. The effects of Chol and DAG are, therefore, not due to specific ones on the enzyme itself, but rather upon the bilayer-organization of the substrate. We discuss the activation of phospholipase D in terms of the influences of DAG and Chol on the structure of hydrophilic region and fluidity of the bilayers.

Magainin 1-induced leakage of entrapped calcein out of negatively-charged lipid vesicles.

Effects of magainin 1, a novel antimicrobial peptide, on the permeability of lipid vesicles were investigated by using calcein as a trapped fluorescent marker. Magainin 1 induces the leakage of calcein specifically out of negatively-charged vesicles. The peptide binds to bovine brain phosphatidylserine sonicated vesicles according to the Langmuir isotherm with a binding constant of 3.8.10(5) M-1 and a binding-site number of 0.10 per lipid molecule. The leakage seems to occur at a critical binding number of approx. 0.03 per lipid molecule. A circular dichroism study revealed that magainin 1 conforms mainly to an unordered structure both in an aqueous solution and in the presence of egg yolk phosphatidylcholine vesicles, whereas to an amphiphilic helix with the phosphatidylserine vesicles. In conclusion, magainin 1 interacts with acidic lipids through electrostatic interactions followed by hydrophobic interactions to form an amphiphilic helix, inducing the leakage.

Inhibition of lipoprotein lipase activity by sphingomyelin: role of membrane surface structure.

We have recently shown that sphingomyelin (SM) strongly inhibits lipoprotein lipase (LPL)-mediated lipolysis in monolayers and emulsion particles. To further evaluate how SM modulates LPL activity on the emulsion surface, the relationship between membrane surface structure and LPL activity was investigated. We measured fluorescence anisotropy of 1-palmitoyl-2-[3-(diphenylhexatrienyl)propionyl]-sn-3-phosphati dylcho line, probing surface acyl chain fluidity, and fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine in H(2)O and D(2)O buffer, assessing the degree of hydration in the head group region. The results revealed that incorporation of egg SM into triolein-egg phosphatidylcholine emulsions markedly increased acyl chain order and decreased head group hydration of the surface monolayers. In contrast, cholesterol was shown to increase head group hydration despite a strong increase in acyl chain order. The close correlation between the apparent K(m) values of LPL and the degree of head group hydration indicated that LPL interacts with the head group region rather than with the hydrophobic interior of the surface monolayers. However, apparent V(max) did not show a simple correlation with any surface structure, and the finding in which SM had no effect on apparent V(max) of medium-chain triglyceride emulsions suggested that the hydrophobic interaction between acyl chains of SM and triglyceride at the emulsion surface is important for determining the apparent V(max). These results showed conclusively that SM inhibits LPL activity mainly by changing the emulsion surface structure and not by a specific interaction between SM and LPL.

A low degree of requirement for Ia-positive macrophages and IL 2 in the induction phase of Listeria monocytogenes-specific T cells in vitro.

We have analyzed the priming process of Listeria-specific T cells using an in vitro primary culture system. Listeria-specific T cells mediating delayed footpad reaction (DFR) and acquired cellular resistance (ACR) upon passive local transfer into naive recipients were generated from non-immune mouse spleen cells when stimulated with viable Listeria monocytogenes primarily in vitro. The effectors were detected on the third day of culture, and culturing for 5 days was sufficient for the generation of effectors mediating the peak level of DFR and ACR. The requirement of T cell subsets, Ia antigen and interleukin 2 (IL 2) for inducing effectors was studied. Presence of macrophages (M phi) and their contact to T cells were required for priming of Listeria-specific T cells in vitro. The presence of Ia antigens on macrophages was absolutely required for priming, but this requirement was lower than that in secondary immune response of Listeria-specific T cells. Effectors could not be generated when L3T4+ cells were depleted, but effectors capable of conferring a full level of DFR and ACR were induced even after the depletion of Lyt2+ cells. Contribution of IL 2 to the generation of effectors during early phase of priming was not observed. IL 2 was not produced in the supernatant of the in vitro primary culture. Precursor cells of the effectors did not respond to exogenously added recombinant IL 2 (rIL 2). Some mechanisms operating in the induction phase of Listeria-specific T cells were clarified in this study.

In vitro primary induction of T cells mediating delayed footpad reaction and acquired cellular resistance to Listeria monocytogenes.

We established an in vitro system generating L. monocytogenes-specific T cells primarily from unprimed spleen cells of mice. Normal spleen cells were cultured for 5 days in the presence of L. monocytogenes in vitro. Viable cells were harvested and assessed for their capacity to confer acquired cellular resistance (ACR) and delayed footpad reaction (DFR) upon local passive transfer to naive syngeneic recipient mice. When normal spleen cells were stimulated with viable L. monocytogenes, the viable cells that were recovered after 5 days of culture conferred a high level of ACR and DFR. Negative selection revealed that the effector cells obtained in primary in vitro culture were Thy 1+, L3T4+, Lyt2- cells. T cells mediating ACR could not be generated in the culture of normal spleen cells with heat-killed bacteria; however, cells mediating only DFR were generated in the presence of a large number of killed L. monocytogenes. The expression of DFR and ACR by T cells generated in this primary culture system was Listeria-specific; reactions were not observed against unrelated bacterial antigens including S. typhimurium, S. aureus, E. coli and PPD. FACS analysis of the cells in culture showed that L3T4+ and Lyt2- T cells were being enriched during culture. The primary generation of antigen-specific T cells in vitro was also possible with spleen cells from NTx mice but not with cells from nude mice, suggesting the presence of Listeria-specific precursors in NTx mice.

Induction of apoptosis in lymphoid tissues of mice after intramuscular injection of enterotoxigenic Escherichia coli enterotoxin.

Although it has been reported that intravenous injection of Escherichia coli enterotoxin induces atrophies of the thymus and spleen by necrosis, the toxin injected intramuscularly to mice induced atrophies of both tissues, which were associated with apoptosis of lymphocytes. Apoptosis predominantly occurred in the thymus and increased in a time-dependent manner up to 26 h and faint ladder band patterns of DNA were observed at 36 h. Although the high dose of toxin also induced in vitro apoptosis in cultured thymocytes, the toxin was not detected in the serum at levels sufficient to cause in vitro apoptosis after intramuscular administration. By flow cytometric analysis, CD4+ CD8+ double-positive T cell and CD45+ positive B cell numbers were found to be mainly decreased in thymus and spleen, respectively, of mice. These results suggest that the atrophies of thymus and spleen by intramuscular administration of the toxin to mice are due to apoptosis of CD4+ CD8+ double-positive T and CD45+ positive B cells, respectively, but the toxin does not reach these cells via the circulation. A different mechanism from that in vitro in cultured cells might be involved in the induction of apoptosis in vivo.

No effect of high-protein food on the stereoselective bioavailability and pharmacokinetics of verapamil.

The effects of high-protein food on the bioavailability of both the racemate and individual enantiomers of verapamil were investigated in 12 healthy volunteers using a randomized crossover design. Food had no effect on any parameter of bioavailability for both the racemate and the individual enantiomers of verapamil except time to maximum concentration (tmax), which was significantly prolonged after food intake. The pharmacokinetics of the enantiomers of norverapamil were not significantly changed by food intake. These results suggest that high-protein food does not alter the pharmacokinetics and bioavailability of either the racemate or the individual enantiomers of verapamil. Therefore, the clinical efficacy of verapamil is not related to food intake, except for a slight prolongation in the time to onset of the pharmacologic effects. The present data can be applied to the high-protein content meal intake.

Low-dose cytarabine and aclarubicin in combination with granulocyte colony-stimulating factor (CAG regimen) for previously treated patients with relapsed or primary resistant acute myelogenous leukemia (AML) and previously untreated elderly patients with AML, secondary AML, and refractory anemia with excess blasts in transformation.

We used the CAG regimen (low-dose cytarabine [10 mg/m2 per 12 hours, days 1-14], aclarubicin [14 mg/m2 per day, days 1-4], and granulocyte colony-stimulating factor [200 micrograms/m2 per day, days 1-14]) for the treatment of patients with primary resistant acute myelogenous leukemia (AML) and previously untreated elderly patients with AML, secondary AML, and refractory anemia with excess blasts in transformation (RAEB-T) in addition to relapsed AML. Forty-three of 69 (62%) patients achieved complete remission (CR), including 29 of 35 (83%) patients with relapsed AML, 1 of 8 patients with primary resistant AML, 5 of 8 elderly patients with previously untreated AML, and 8 of 18 patients with previously untreated secondary AML or RAEB-T. Ten of 22 (45%) patients > or = 65 years old achieved CR. The patients who achieved CR received at least 1 course of modified CAG therapy as the first consolidation therapy, followed by various second consolidation and intensification therapies. The median disease-free survival and overall survival were 8 and 15 months, respectively, for relapsed AML; 11 and 8 months for the elderly patients; and 8 and 17 months for secondary AML and RAEB-T. Myelosuppression was mild to moderate, and other than fever, severe nonhematologic toxicity was rare. CAG as the induction therapy seems promising for the treatment of various categories of poor-prognosis AML.

Evaluation of particulate embolic materials with MR imaging, scanning electron microscopy, and phase-contrast microscopy.

PURPOSE: To analyze the properties and embolic effect of microfibrillar collagen (MFC), Gelfoam powder, and polyvinyl alcohol (PVA) materials that are used in embolization procedures in the head and neck. METHODS: The shape and surface of these embolic agents were examined with scanning electron microscopy and phase-contrast microscopy. The mean number of areas of T2-weighted high signal intensity was measured on MR images in a rat embolization model to estimate the embolic effect. RESULTS: By scanning electron microscopy and phase-contrast microscopy, MFC appears fibriform and has various sizes and an irregular surface. Gelfoam is of uniform size and has a smooth surface. PVA materials are granulated and have a rough surface. MFC is somewhat suspendable and its shape changes moderately after suspension. Gelfoam is very suspendable and its shape changes rapidly. PVA showed only mild swelling. The embolic effect of MFC was the lowest of the materials examined. Large PVA particles (250 to 500 microns) showed a lesser embolic effect than Gelfoam or small PVA particles (50 to 150 microns) or medium-sized PVA particles (150 to 250 microns). No significant differences were observed among the embolic effects of Gelfoam, small PVA particles (50 to 150 microns), and medium PVA particles (150 to 250 microns). CONCLUSIONS: MFC and large PVA particles (250 to 500 microns) should be used for embolization of vascular anatomy involving potentially dangerous anastomoses. Gelfoam, PVA particles of 150- to 250-micron diameter, and PVA particles of 50- to 150-micron diameter are adequate for embolization involving homogeneous and peripheral anatomy.

Resonance energy transfer study of peptide-lipid complexes.

Resonance energy transfer involving tryptophan as a donor and anthrylvinyl-labeled phosphatidylcholine (AV-PC), 3-methoxybenzanthrone (MBA) and 8-anilino-1-naphthalene sulfonic acid (ANS) as acceptors has been examined to obtain information on the structure of peptide-lipid systems consisting of 18A or Ac-18A-NH(2) peptides and large unilamellar phosphatidylcholine vesicles. The lower and upper limits for the tryptophan distance from the bilayer midplane have been assessed in terms of the models of energy transfer in two-dimensional systems, taking into account orientational effects. Evidence for the existence of preferential orientations of Ac-18A-NH(2) with respect to the lipid-water interface has been obtained.