A New Approach to Quantify and Grade Radiation Dermatitis Using Deep-Learning Segmentation in Skin Photographs.

AIMS: Objective evaluation of radiation dermatitis is important for analysing the correlation between the severity of radiation dermatitis and dose distribution in clinical practice and for reliable reporting in clinical trials. We developed a novel radiation dermatitis segmentation system based on convolutional neural networks (CNNs) to consistently evaluate radiation dermatitis. MATERIALS AND METHODS: The radiation dermatitis segmentation system is designed to segment the radiation dermatitis occurrence area using skin photographs and skin-dose distribution. A CNN architecture with a dilated convolution layer and skip connection was designed to estimate the radiation dermatitis area. Seventy-three skin photographs obtained from patients undergoing radiotherapy were collected for training and testing. The ground truth of radiation dermatitis segmentation is manually delineated from the skin photograph by an experienced radiation oncologist and medical physicist. We converted the skin photographs to RGB (red-green-blue) and CIELAB (lightness (L∗), red-green (a∗) and blue-yellow (b∗)) colour information and trained the network to segment faint and severe radiation dermatitis using three different input combinations: RGB, RGB + CIELAB (RGBLAB) and RGB + CIELAB + skin-dose distribution (RGBLAB_D). The proposed system was evaluated using the Dice similarity coefficient (DSC), sensitivity, specificity and normalised Matthews correlation coefficient (nMCC). A paired t-test was used to compare the results of different segmentation performances. RESULTS: Optimal data composition was observed in the network trained for radiation dermatitis segmentation using skin photographs and skin-dose distribution. The average DSC, sensitivity, specificity and nMCC values of RGBLAB_D were 0.62, 0.61, 0.91 and 0.77, respectively, in faint radiation dermatitis, and 0.69, 0.78, 0.96 and 0.83, respectively, in severe radiation dermatitis. CONCLUSION: Our study showed that CNN-based radiation dermatitis segmentation in skin photographs of patients undergoing radiotherapy can describe radiation dermatitis severity and pattern. Our study could aid in objectifying the radiation dermatitis grading and analysing the reliable correlation between dosimetric factors and the morphology of radiation dermatitis.

Human CD4+ CD39+ regulatory T cells produce adenosine upon co-expression of surface CD73 or contact with CD73+ exosomes or CD73+ cells.

While murine CD4(+) CD39(+) regulatory T cells (T(reg)) co-express CD73 and hydrolyze exogenous (e) adenosine triphosphate (ATP) to immunosuppressive adenosine (ADO), surface co-expression of CD73 on human circulating CD4(+) CD39(+) T(reg) is rare. Therefore, the ability of human T(reg) to produce and utilize ADO for suppression remains unclear. Using mass spectrometry, we measured nucleoside production by subsets of human CD4(+) CD39(+) and CD4(+) CD39(-)CD73(+) T cells or CD19(+) B cells isolated from blood of 30 volunteers and 14 cancer patients. CD39 and CD73 expression was evaluated by flow cytometry, Western blots, confocal microscopy or reverse transcription-polymerase chain reaction (RT-PCR). Circulating CD4(+) CD39(+) T(reg) which hydrolyzed eATP to 5'-AMP contained few intracytoplasmic granules and had low CD73 mRNA levels. Only ∼1% of these T(reg) were CD39(+) CD73(+) . In contrast, CD4(+) CD39(neg) CD73(+) T cells contained numerous CD73(+) granules in the cytoplasm and strongly expressed surface CD73. In vitro-generated T(reg) (Tr1) and most B cells were CD39(+) CD73(+) . All these CD73(+) T cell subsets and B cells hydrolyzed 5'-AMP to ADO. Exosomes isolated from plasma of normal control (NC) or cancer patients carried enzymatically active CD39 and CD73(+) and, when supplied with eATP, hydrolyzed it to ADO. Only CD4(+) CD39(+) T(reg) co-incubated with CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes produced ADO. Thus, contact with membrane-tethered CD73 was sufficient for ADO production by CD4(+) CD39(+) T(reg). In microenvironments containing CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes, CD73 is readily available to CD4(+) CD39(+) CD73(neg) T(reg) for the production of immunosuppressive ADO.

Allergenicity of recombinant profilins from Japanese hop, Humulus japonicus.

BACKGROUND AND OBJECTIVE: Pollen from Japanese hop, Humulusjaponicus, is a major cause of pollinosis in Korea. Profilin (15 kDa) from Humulus scandens has been associated with strong allergenicity in allergic Chinese patients. Profilin has also been detected in pollen extract from Korean Japanese hop by proteomic analysis and immunoglobulin (Ig) E immunoblotting. However, the allergenicity of allergens isolated from Japanese hop has not been investigated in Korean individuals. This study was undertaken to produce recombinant profilin from Japanese hop and evaluate its allergenicity. METHODS: Complementary DNA sequences encoding 2 isoallergens were cloned by reverse transcription polymerase chain reaction and their recombinant proteins expressed in Escherichia coli. The IgE-binding reactivities of the recombinant allergens were assessed by enzyme-linked immunosorbent assay. RESULTS: The deduced amino acid sequences of the H. japonicus profilins were 68.7% to 80.2% homologous with profilins from mugwort (Art v 4), ragweed (Amb a 14), and birch (Bet v 2). Two isoallergens of profilin from H. japonicus were 78.2% identical. Notably, the cDNA sequences of these 2 isoallergens were 98.5% (AY268422) and 98.7% (AY268424) identical to those of H. scandens. Serum samples from Japanese hop-sensitized individuals showed 12.9% IgE reactivity to both of the recombinant profilin isoallergens from H. japonicus, indicating that profilin may not be an allergenically dominant component of Japanese hop pollen. The recombinant profilins showed only 0% to 9.3% inhibition of the crude extract. CONCLUSIONS: Two isoallergens of profilin that are highly conserved with those of mugwort, ragweed, and birch were identified in H. japonicus. Profilins from Japanese hop pollen may play a minor role in the pathogenesis of pollinosis in Koreans.

YKL-40 in induced sputum after allergen bronchial provocation in atopic asthma.

BACKGROUND: Serum chitinase-like proteins such as YKL-40 in asthmatic patients are known to positively correlate with disease severity but controversy remains regarding their role. The allergen bronchial provocation test (ABPT) can induce allergic airway inflammation in individuals with atopic asthma. OBJECTIVE: To evaluate the induction and kinetics of YKL-40 during allergen-induced airway inflammation in atopic asthmatics. METHODS: Thirteen patients were enrolled from May to November 2008. They all underwent ABPT with Dermatophagoides farinae crude extract. Induced sputums (IS) and serum were collected 3 times: 7 days before ABPT (baseline), 7 hours after ABPT, and 24 hours after ABPT. We examined the cytology of induced sputum (IS) and measured levels of YKL-40, interleukin (IL) 4, IL-5, IL-13, IL-33, tumor necrosis factor (TNF) alpha, and eosinophilic cationic protein (ECP) in IS and/or serum. RESULTS: Following ABPT, total inflammatory cells, eosinophils, and neutrophils increased in a time-dependent manner in IS. YKL-40 levels were increased in IS but not in serum at 7 or 24 hours after ABPT (P=.011 and P=.006, respectively). Similarly to YKL-40, IL-5 and ECP levels were also increased in IS at 7 and 24 hours after ABPT (P=.011 for IL-5 and P=.006 for ECP). Overall, YKL-40 levels were well correlated with ECP levels in IS (p=0.576, P<.001). CONCLUSIONS: YKL-40 levels increased immediately in IS but not in the serum of atopic asthmatics. The correlation between YKL-40 levels and ECP in IS suggests that YKL-40 may play a pathophysiologic role in human atopic asthma.

Ectopic matrix metalloproteinase-9 expression in human brain tumor cells enhances oncolytic HSV vector infection.

Oncolytic herpes simplex virus (oHSV) vectors have shown promise in the treatment of patients with recurrent brain tumors although few complete responses have accrued. Impediments to effective therapy include limited vector distribution on delivery, a consequence of injected virion particle trapping in the tumor extracellular matrix (ECM). To enhance virus delivery and spread, we investigated the use of the matrix metalloproteinase-9 (MMP-9) as a means to degrade collagen type IV, a major component of the ECM and basement membranes of gliomas that is absent in normal brain tissue. SK-N-AS neuroblastoma cells were transduced for constitutive, elevated expression of MMP-9, which did not enhance tumor cell migration in vitro or tumor progression in a murine xenograft brain tumor model. MMP-9 expression improved the distribution and infection of oHSV vectors in spheroid model in vitro. Furthermore, MMP9 induced a vector infection over larger areas of brain tumors in vivo. These results suggest that vector delivery and distribution in vivo can be improved by compromising the ECM, potentially enhancing oncolytic efficacy.

Exercise-induced airway obstruction in young asthmatics measured by impulse oscillometry.

BACKGROUND: Impulse oscillometry (IOS) is a good method for measuring airway resistance. It does not require special breathing skills and it can reflect different aspects of airway obstruction to those revealed by spirometry, which is an effort-dependent maneuver. OBJECTIVE: To evaluate the characteristics of airway obstruction in young asthmatics after an exercise bronchial provocation test (EBPT) using IOS. METHODS: Forty-seven young adults were enrolled in the study. All the participants underwent a methacholine bronchial provocation test (MBPT) and an EBPT for the evaluation of their asthma. IOS and spirometric parameters were collected at baseline and at 0, 5, 10, 20, and 30 minutes post-EBPT.The participants were divided into 2 groups according to MBPT positivity: an airway hyperresponsiveness (AHR) group and a no-AHR group. RESULTS: There were differences in the percent decrease in forced expiratory volume in the first second (FEV1) between the 2 groups at 5, 10, and 20 minutes after exercise. Resistance at 5 Hz (R5) increased in the AHR group but not in the no-AHR group at 5 and 10 minutes after exercise. Integration of reactance from 5 Hz to resonance frequency (area of reactance, AX) was also increased in the AHR group at only 5 and 10 minutes post-EBPT. Delta R5 and delta AX at 5 and 10 minutes post-exercise were well correlated with the percent decrease in FEV1. CONCLUSIONS: IOS parameters, especially delta R5 and delta AX, may be useful for performing objective evaluations and improving our understanding of exercise-induced airway obstruction in young asthmatics.

Alpha-T-catenin (CTNNA3) gene was identified as a risk variant for toluene diisocyanate-induced asthma by genome-wide association analysis.

BACKGROUND: Toluene diisocyanate (TDI) is the most important cause of occupational asthma, but the genetic mechanism of TDI-induced asthma is still unknown. OBJECTIVE: The objective of the study was to identify susceptibility alleles associated with the TDI-induced asthma phenotype. METHODS: We conducted a genome-wide association study in 84 patients with TDI-induced asthma and 263 unexposed healthy normal controls using Affymetrix 500K SNPchip. We also investigated the relationships between genetic polymorphisms and transcript levels in Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with TDI-induced asthma enrolled in this study. RESULTS: Genetic polymorphisms of CTNNA3 (catenin alpha 3, alpha-T catenin) were significantly associated with the TDI-induced asthma phenotype (5.84 x 10(-6) for rs10762058, 1.41 x 10(-5) for rs7088181, 2.03 x 10(-5) for rs4378283). Carriers with the minor haplotype, HT2 [GG], of two genetic polymorphisms (rs10762058 and rs7088181) showed significantly lower PC(20) methacholine level (P=0.041) and lower mRNA expression of CTNNA3 than non-carriers (P=0.040). A genetic polymorphism in the 3' downstream region of CTNNA3 (rs1786929), as identified by DNA direct sequencing, was significantly associated with the TDI-induced asthma phenotype (P=0.015 in recessive analysis model) and the prevalence of serum-specific IgG to cytokeratin 19 (P=0.031). CONCLUSION: These findings suggested that multiple genetic polymorphisms of CTNNA3 may be determinants of susceptibility to TDI-induced asthma.

Characterization of the major allergens of Pachycondyla chinensis in ant sting anaphylaxis patients.

BACKGROUND: The ant species Pachycondyla chinensis, which has spread from Far Eastern Asia to New Zealand and North America, induces anaphylactic reactions in human with its sting. However, the major allergens of P. chinensis have not yet been characterized. METHODS: We selected seven patients with histories of anaphylaxis induced by P. chinensis. Two-dimensional electrophoresis (2-DE) was used to identify the major allergens. We subsequently performed Western blots for P. chinensis-specific IgEs, N-terminal amino acid sequencing, ESI-MS/MS, and RT-PCR using primers based on the N-terminal sequence. RESULTS: Six of the anaphylactic subjects had an IgE specific to a 23 kDa allergen of P. chinensis. Two candidates for major allergens, 23 kDa (pI 8.7) and 25 kDa (pI 6.2), were revealed by 2-DE using P. chinensis-specific IgE immunoblotting. In N-terminal sequencing and ESI-MS/MS analysis, 23 kDa (pI 8.7) and 25 kDa (pI 6.2) allergens, belonging to the protein families of antigen 5, were identified and share marked amino acid sequence similarity. The 23 kDa allergen is 206 amino acids in length and homology searches showed 54.0% and 50.0% homology with Sol i 3 and Ves v 5, respectively. CONCLUSION: The major allergens of P. chinensis are 23 kDa (pI 8.7) and 25 kDa (pI 6.2) proteins that belong to the antigen 5 family of proteins.

Relationship between neurokinin 2 receptor gene polymorphisms and serum vascular endothelial growth factor levels in patients with toluene diisocyanate-induced asthma.

BACKGROUND: Among the various pathogenic mechanisms of toluene diisocyanate (TDI)-induced asthma, a contribution from neurogenic inflammation has been suggested. OBJECTIVE: To evaluate neurokinin 2 receptor (NK2R) gene polymorphisms in association with the clinical phenotype of TDI-induced asthma, 70 TDI-induced occupational asthma (TDI-OA)patients, 59 asymptomatic exposed controls (AEC), and 93 unexposed healthy controls (NC) were enrolled in the study. METHODS: Two single-nucleotide polymorphisms (SNPs) of NK2R, 7853G>A (Gly231Glu) and 11 424G>A (Arg375His), were genotyped using a single base extension method. The levels of PC20 methacholine, specific IgE and IgG to TDI-human serum albumin conjugate, and serum vascular endothelial growth factor (VEGF), matrix metalloproteinase-9, and TGF-beta1 were compared according to the NK2R genotypes of the subjects with TDI-OA and AEC. RESULTS: No significant differences in allele, genotype, or haplotype frequencies of these two SNPs were noted among the three groups (P>0.05, respectively). Moreover, subjects with the NK2R 7853GG genotype had higher serum VEGF levels than those with GA or AA among the TDI-exposed workers (P=0.040). CONCLUSION: The NK2R 7853GG genotype may contribute to increased serum VEGF levels, which result in airway inflammation after TDI exposure.

HLA DRB1*15-DPB1*05 haplotype: a susceptible gene marker for isocyanate-induced occupational asthma?

BACKGROUND: There has been no study for evaluating the associations of human leukocyte antigen (HLA) class I and II alleles with toluene diisocyanate (TDI)-induced asthma in an Asian population. OBJECTIVE: The aim of this study was to investigate a susceptible or protective marker of HLA class I and II alleles in TDI-induced asthma. METHODS: Fifty-five patients with TDI-induced asthma patients (group I) showing positive responses on TDI bronchoprovocation test, 47 asymptomatic exposed subjects (group II) and 95 unexposed healthy nonatopic controls (group III) were enrolled in our study. HLA class I and II genotyping was done by the direct DNA sequencing method. RESULTS: The allelic frequency of C*09 (15.5%) was significantly higher in group I than in group III (6.8%, P = 0.019), but this statistical significance disappeared after correction was made for multiple comparisons. On two-locus and three-locus haplotype analysis, the allelic frequency of HLA DRB1*15-DPB1*05 in group I (10.6%) was significantly higher than that of group II (0%, P = 0.001) and group III (2.5%, P = 0.003). The allelic frequencies of HLA A*02-DRB1*15, A*02-DQB1*06, B*62-C*09 and A*02-DRB1*15-DQB1*06 were significantly higher in group I (8.5%, 10.3%, 8.2% and 6.8%, respectively) than those allelic frequencies of group III (1.3%, P = 0.002; 1.6%, P = 0.001; 0.6%, P < 0.0001; 0%, P < 0.0001, respectively). The allelic frequencies of HLA DQB1*06-DPB1*05 and DRB1*15-DQB1*06-DPB1*05 were significantly higher in group I (16.0% and 10.5%) than those in group II (2.5%, P = 0.001; 0%, P = 0.001), while the frequencies of DRB1*09-DPB1*05 and DRB1*09-DQB1*0303-DPB1*05 were significantly lower in group I (0% and 0%) than those of group II (7.4%, P = 0.004; 7.5%, P = 0.004). These differences remained statistically significant even after the correction for multiple comparisons. CONCLUSIONS: The HLA haplotype DRB1*15-DPB1*05 can be a susceptibility gene marker for the development of TDI-induced asthma among the exposed workers in the Korean population.

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-beta peptide in vivo.

Accumulation of insoluble aggregates of amyloid-beta peptide (Abeta), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer's disease (AD). Consequently, downregulation of APP, or enhanced clearance of Abeta, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Abeta accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Abeta (HSV-neprilysin), substantially inhibited accumulation of Abeta. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APP(Sw)), resulted in rapid Abeta accumulation. Co-inoculation of LV-APP(Sw) with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Abeta accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Abeta accumulation in the brain, thus providing useful tools to clarify the role of Abeta in AD that may facilitate the development of novel therapies for this important disease.

Allergen-specific immunosuppression by ovalbumin fused with diphtheria toxin in mice sensitized with albumins of different origin.

BACKGROUND: We previously reported that ovalbumin-diphtheria toxin (OVA-DT) fusion protein eliminates mast cells bearing OVA-specific IgE and protects OVA-sensitized mice from fatal anaphylaxis induced by OVA challenge. OBJECTIVE: To prove the specificity of therapeutic effect of OVA-DT to allergy induced by OVA only and not by other allergens such as human serum albumin (HSA), and to examine the cytotoxic effect of OVA-DT on B cells bearing OVA-specific IgE. METHODS: Mice were sensitized with two different antigens, OVA and HSA, and then treated with OVA-DT. The therapeutic effect of OVA-DT on the allergy response to each of allergen was evaluated by anaphylactic test. The effect of OVA-DT on the production of allergen-specific Ig isotypes of the sensitized mice and the cytotoxic effect of OVA-DT on B cells expressing OVA-specific IgE were examined. RESULTS: OVA-DT suppressed only OVA-induced allergy but not HSA-induced allergy in mice sensitized with a mixture of OVA and HSA. The suppression was prolonged even to the mice boosted with the same allergen 14 days after last treatment of OVA-DT. In addition, when the sensitized mice were boosted with the same allergens 14 days after last treatment of OVA-DT, the mice showed to increase the production of OVA-specific IgG2a/IgG3 and decreased that of OVA-specific IgE. OVA-DT targeted B cells bearing OVA-specific IgE, and killed them by DT-mediated cytotoxicity. CONCLUSION: The therapeutic effect of OVA-DT was specific to OVA-induced allergy and the suppression of OVA-induced allergy was continuously shown in the mice boosted with the same allergens. This is considered to be caused by the increase of OVA-specific IgG2a and IgG3, and because of the decrease of OVA-specific IgE by killing of B cells bearing OVA-specific IgE.

Ultrafast photoinduced reflectivity transients in doped manganite.

The temperature and magnetic field dependence of ultrafast photoinduced spin and quasiparticle relaxation dynamics is reported in La(0.67)Ca(0.33)MnO(3) and LaMnO(3) single crystals and thin films. Both manganites reveal an unusually slow ( approximately 10 micros) carrier relaxation process attributed to the spin-lattice relaxation in localized states. The quasiparticle dynamics is governed by the temperature- and magnetic field-dependent pseudogap in La(0.67)Ca(0.33)MnO(3), and by the temperature-independent Jahn-Teller gap in LaMnO(3). The loss of spectral weight near the Fermi level in La(0.67)Ca(0.33)MnO(3) strongy affects the quasiparticle relaxation dynamics as temperature increases from below T(C). Our results show that the coupled dynamics of charge, spin and lattice is strongly correlated with the distinct gap structures in these manganites.

Allergenicity of recombinant Bla g 7, German cockroach tropomyosin.

BACKGROUND: Cockroach infestation may sensitize and elicit allergic responses to genetically predisposed individuals. Invertebrate tropomyosins are a frequent cause of allergy and highly cross-reactive in nature. In this study, we aimed to produce recombinant German cockroach tropomyosin and investigate its allergenicity. METHODS: German cockroach tropomyosin (Bla g 7) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The cloned cDNA was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-nitrilotriacetic (NTA) acid resin. The allergenicity of the recombinant tropomyosin was examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The cloned Bla g 7 shared up to 91% amino acid sequence identity with other cockroach tropomyosins. ELISA showed a recombinant Bla g 7 sensitization rate of 16.2% to German cockroach allergic sera. Recombinant tropomyosin was able to inhibit 32.4% of the specific IgE binding to cockroach extract. CONCLUSIONS: Tropomyosin represents a minor allergen in cockroach extracts. It is hoped that recombinant tropomyosin will be useful for further studies and clinical applications.

Trace organic contaminants, including toxaphene and trifluralin, in cotton field soils from Georgia and South Carolina, USA.

Residues of organic contaminants--including toxaphene, DDT, trifluralin, hexachlorocyclohexanes, polychlorinated biphenyls, polycyclic aromatic hydrocarbons (PAHs) and nonylphenol--were measured in 32 cotton field soils collected from South Carolina and Georgia in 1999. Toxaphene, trifluralin, DDT and PAHs were the major contaminants found in these soils. The maximum concentration of toxaphene measured was 2,500 ng/g dry weight. Trifluralin was detected in all the soils at concentrations ranging from 1 to 548 ng/g dry weight. Pesticide residues were not proportional to soil organic carbon content, indicating that their concentrations were a reflection of application history and dissipation rates rather than air-soil equilibrium. Soil extracts were also subjected to in vitro bioassays to assess dioxinlike, estrogenic, and androgenic/glucocorticoid potencies. Relatively more polar fractions of the soils elicited estrogenic and androgenic/glucocorticoid activities, but the magnitude of response was much less than those found in coastal marine sediments from industrialized locations.

Der p 2 isoallergens have different allergenicity, and quantification with 2-site ELISA using monoclonal antibodies is influenced by the isoallergens.

BACKGROUND: Der p 2 isoallergens have been reported and the possibility of different allergenicity has also been suggested. In addition, the quantification with 2-site ELISA may be affected by the isoallergens. OBJECTIVES: Two different recombinant Der p 2 (rDer p 2) isoallergens were compared in terms of human IgE responses and the reliability of quantification of them with two-site ELISA kits which use monoclonal antibodies (mAbs) as capture and detection of Der p 2. METHODS: Seven different Der p 2 cDNA from the cultured Dermatophagoides pteronyssinus (DP) were cloned and polymorphism in nine amino acid residues was found. Two different recombinant isoallergens (rDer p 2A and rDer p 2B) were expressed and compared to their human IgE immune responses by ELISA and the ELISA inhibition test with 23 sera of DP-allergic patients. The reliability of quantification of two different available 2-site ELISA kits, which used mAbs for capture and detection of Der p 2, was evaluated. RESULTS: The ELISA optical density of rDer p 2B-specific IgE (sIgE) was higher than that of rDer p 2A (P < 0.001). The ELISA inhibition curve of rDer p 2B sIgE in pool I sera (n = 5; high sIgE both to rDer p 2A and rDer p 2B) did not show any differences in the 50% inhibition concentration and maximum inhibitory percentage of rDer p 2A and rDer p 2B sIgE. However, with pool II sera (n = 5; markedly higher sIgE to rDer p 2B than rDer p 2A), the 50% inhibitory concentrations (10 microg/mL vs. 40 ng/mL) and maximum inhibitory percentage (61% vs. 99%) of rDer p 2B sIgE with the two recombinant isoallergens were quite different. rDer p 2B could be quantified with two different 2-site ELISA kits, but rDer p 2A was detected by only one kit. CONCLUSION: We conclude that isoallergens of Der p 2 may have different IgE immune responses. Quantification of Der p 2 with 2-site ELISA kits that adopted mAbs, might be affected by the prevalent form of the isoallergens in reservoir dust.

Localization of Der f 2 in the gut and fecal pellets of Dermatophagoides farinae.

BACKGROUND: House dust mite derived materials are known to be the most potent agent inducing allergic diseases. Localization of Der f 2 was attempted to specify the sites and concentrations of Der f 2 within the mite, which may indicate the importance of secreted materials and nonexcreted body components as allergen sources. METHODS: Serial cryostat sections of embedded live mites and the fecal pellets, collected by brush, were immunoprobed using monoclonal antibody (mAb) 2F38 raised against recombinant (r) Der f 2. RESULTS: Highest concentrations were found in the anterior midgut, implying that this is the site of Der f 2 synthesis and secretion. Digestive material and defecated fecal pellets were also labeled with mAb. CONCLUSIONS: Our results show that the major allergen, Der f 2, found in the house dust mite D. farinae is derived from the digestive tract, and is concentrated in the feces.

Monoclonal antibodies to recombinant Der f 2 and development of a two-site ELISA sensitive to major Der f 2 isoallergen in Korea.

BACKGROUND: Der f 2 is a major sensitizing allergen in patients allergic to house dust mites worldwide. Isoforms of Der f 2 have been reported and are known to have different antigenicities. The aim of this study was to facilitate antigenic analysis and to develop an improved method for the detection of Der f 2 isoallergen, which is prevalent in Korea. METHODS: A two-site ELISA was developed with monoclonal antibodies (mAbs) which were produced against recombinant Der f 2 (rDer f 2) and applied to assess Der f 2 in bedding samples. RESULTS: A major isoform of Der f 2, found in Korea, was found to have amino acid variations especially at position 100 from lysine to glutamic acid, which is known to reduce significantly the binding affinity of mAbs when used to assess group 2 allergens. The detection limit of the developed two-site ELISA was determined to be about 8 ng/ml with rDer f 2 and 1 microg/ml with Derntatophagoides farinae crude extract. The average amount of Der f 2 in dust obtained from bedding samples from 89 homes in Seoul was estimated to be 25.61+/-10.70 microg/g dust. CONCLUSIONS: Assays using mAbs for rDer f 2 could be useful for the assessment of environmental allergen exposure and mAbs could be used to further characterize the isoallergens of Der f 2.

Molecular cloning and characterization of mouse cardiac junctate isoforms.

Junctate is a newly identified integral ER/SR membrane calcium binding protein, which is an alternative splicing form of the same gene generating aspartyl beta-hydroxylase and junctin. Screening a mouse heart cDNA library using canine junctin cDNA as a probe yielded three complete mouse heart cDNAs. One of the cDNAs is homologous to the previously reported human junctate. The three mouse junctate proteins are composed of 270, 259, and 215 amino acids (we named them junctate-1, -2, and -3). The apparent molecular masses of the mouse junctates in SDS-PAGE were in the range between 40 and 53 kDa. Northern and Western blot analyses indicate that mouse junctates are expressed in heart, brain, spleen, lung, liver, kidney, and stomach, but not in skeletal muscle. The apparent molecular weights of junctates from heart and brain were somewhat different from those from the other tissues tested, suggesting that there are tissue-specific expression patterns of the different junctate isoforms. Immunohistochemical studies showed that junctates were expressed both in ventricular and atrial tissues. This is the first study that shows the presence of 3 distinct cardiac junctate isoforms expressed in various mammalian tissues.