Formation of IgE-Allergen-CD23 Complex Changes in Children Treated with Subcutaneous Immunotherapy for Japanese Cedar Pollinosis.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is used to treat Japanese cedar (JC) pollinosis. The formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis has not yet been fully elucidated. OBJECTIVE: The objective of this study was to investigate the formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis. METHODS: Eleven patients were treated with 3-year SCIT for JC pollinosis at Sa-gamihara National Hospital from 2013 to 2014. Nasal and ocular symptoms (in terms of symptom scores) during the scattering of JC pollen and immunological changes were investigated. Levels of JC pollen-specific antibodies (IgE and IgG4) were measured by ImmunoCAP assays. To detect the changes in allergen-presenting ability of B cells, the levels of IgE-allergen-CD23 complexes in serum were measured by a cell-free, enzyme-linked immunosorbent-facilitated antigen-binding assay. RESULTS: The median (interquartile range) age of the subjects was 8 (6-10) years. Three patients (27%) had comorbid atopic dermatitis, and 5 patients (45%) had comorbid bronchial asthma. Before starting SCIT, the total IgE level was 373 (75-2,870) kU/L, and the level of JC pollen-specific IgE was 77.2 (15.4-528) kUA/L. Symptom scores improved significantly from the year after treatment. JC pollen-specific IgE levels did not change after 3 years of treatment. JC pollen-specific IgG4 levels increased significantly throughout the treatment period. The levels of IgE-allergen-CD23 complexes decreased significantly after 3 years of treatment. CONCLUSION: The ability of IgE-allergen complexes to bind to CD23 decreased after SCIT, suggesting that increasing levels of IgE-blocking antibodies, including IgG4, may play an important role in the mechanism of SCIT.

Long-term treatment of Japanese cedar pollinosis with Japanese cedar pollen SLIT drops and persistence of treatment effect: A post-marketing clinical trial.

BACKGROUND: There have been no reports of treatment effect persistence after long-term sublingual immunotherapy (SLIT) in patients with Japanese cedar (JC) pollinosis. Therefore, we conducted a post-marketing clinical trial to investigate the efficacy, safety, and effect persistence of JC pollen SLIT drops after approximately 3 years of treatment. METHODS: This was an open-label trial of 233 patients with JC pollinosis who were treated with JC pollen SLIT drops for approximately 3 years (2015-2017) and followed-up for an additional 2 years (2018-2019). Efficacy and effect persistence were evaluated using nasal and ocular symptom scores, daily use of rescue medication, and Japanese Rhinoconjunctivitis Quality of Life Questionnaire scores recorded during the JC pollen dispersal season of each year. Safety was evaluated by monitoring adverse events and adverse drug reactions. RESULTS: The mean combined total nasal symptom and medication score (range 0-18) during the peak symptom periods of 2015 through 2019 were 5.47 ± 3.38, 4.52 ± 3.13, 3.58 ± 2.63, 5.28 ± 4.01, and 6.83 ± 4.65, respectively. The percentage of patients who used no rescue medications during the same periods was 64.8%, 75.2%, 80.3%, 63.7%, and 50.3%, respectively. A total of 138 adverse drug reaction incidents were recorded in 73 of the 233 patients (31.3%), of which 134 incidents (97.1%) were mild in severity. CONCLUSIONS: JC pollen SLIT drops demonstrated treatment duration-dependent efficacy with effects that persisted for 2 years after cessation of treatment. The drug had a favorable safety profile over the 5-year study period.

[EFFICACY, SAFETY AND IMMUNOLOGICAL RESPONSE WITH SQ HOUSE DUST MITE SUBLINGUAL IMMUNOTHERAPY-TABLET BY BODY WEIGHT IN CHILDREN].

BACKGROUND: Sublingual immunotherapy-tablet (SLIT-tablet) treatment includes the same dose regardless of the patients' age or body weight. We investigated the efficacy, safety and immunological response of SQ house dust mite (HDM) SLIT-tablet treatment in relation to body weight in children. METHODS: Total combined rhinitis score (TCRS), adverse events (AEs), adverse drug reactions (ADRs) and immunological response (IgE, IgG4) were evaluated post hoc in three subgroups (body weight < 30kg, 30-44kg, ≥ 45kg) of patients from a clinical trial for Japanese children with HDM allergic rhinitis (JapicCTI-152953). RESULTS: No apparent differences in TCRS were observed between the three subgroups. No differences in the frequency or nature of AEs were detected between the subgroups but the incidence of ADRs was decreased in the lower body weight subgroup. The most common ADRs occurred locally in the oral cavity and were categorized as mild. The levels of HDM specific IgE and IgG4 were increased compared to baseline in all subgroups. CONCLUSION: There were no influences of body weight for efficacy, safety, and immunological response in treatment with SQ HDM SLIT-tablet. These results indicated that SLIT dosage in children is same as adults without any concern in safety.

Association of the winged helix motif of the TFIIEα subunit of TFIIE with either the TFIIEß subunit or TFIIB distinguishes its functions in transcription.

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and ß, and plays essential roles in transcription. Structure-function studies indicate that TFIIE has three-winged helix (WH) motifs, with one in TFIIEα and two in TFIIEß. Recent studies suggested that, by binding to the clamp region of RNA polymerase II, TFIIEα-WH promotes the conformational change that transforms the promoter-bound inactive preinitiation complex to the active complex. Here, to elucidate its roles in transcription, functional analyses of point-mutated human TFIIEα-WH proteins were carried out. In vitro transcription analyses identified two classes of mutants. One class was defective in transcription initiation, and the other was defective in the transition from initiation to elongation. Analyses of the binding of this motif to other general transcription factors showed that the former class was defective in binding to the basic helix-loop-helix motif of TFIIEß and the latter class was defective in binding to the N-terminal cyclin homology region of TFIIB. Furthermore, TFIIEα-WH bound to the TFIIH XPB subunit at a third distinct region. Therefore, these results provide further insights into the mechanisms underlying RNA polymerase II activation at the initial stages of transcription.

Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes.

Programmed cell death can be divided into several categories including type I (apoptosis) and type II (autophagic death). The Bcl-2 family of proteins are well-characterized regulators of apoptosis, and the multidomain pro-apoptotic members of this family, such as Bax and Bak, act as a mitochondrial gateway where a variety of apoptotic signals converge. Although embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, we found that these cells still underwent a non-apoptotic death after death stimulation. Electron microscopic and biochemical studies revealed that double knockout cell death was associated with autophagosomes/autolysosomes. This non-apoptotic death of double knockout cells was suppressed by inhibitors of autophagy, including 3-methyl adenine, was dependent on autophagic proteins APG5 and Beclin 1 (capable of binding to Bcl-2/Bcl-x(L)), and was also modulated by Bcl-x(L). These results indicate that the Bcl-2 family of proteins not only regulates apoptosis, but also controls non-apoptotic programmed cell death that depends on the autophagy genes.

[Pharmacological and clinical study results of Berotralstat Hydrochloride for long-term prophylactic treatment of hereditary angioedema].

Hereditary angioedema (HAE) is a rare disease that causes serious health problem and affects on quality of life for patient due to recurrent episodes of angioedema in various body such as the skin, larynx, digestive tract, and limbs. Many HAE patients have deficiency or dysfunction of C1 inhibitor, impaired regulation of plasma kallikrein activity and overproduction of bradykinin, resulting in leading to episodes of increased capillary hyper permeability and angioedema. Therapy of HAE consists of on-demand treatment for acute attack and prophylactic treatment by suppressing the onset of acute attack in the short and long term. However, no drug has been approved for long-term prophylaxis in Japan. Berotralstat hydrochloride (ORLADEYO Capsules 150 mg) is an oral, selective plasma kallikrein inhibitor approved for the suppression of the onset of acute attacks in HAE in Japan in January 2021. Preclinical studies demonstrated that Berotralstat is a potent and highly specific inhibitor of human plasma kallikrein activity. Berotralstat suppressed bradykinin production in the HUVEC system. Clinical studies demonstrated that oral administration of Berotralstat to HAE type I or type II patients at a dose of 150 mg once daily showed a reduction of HAE attack rate and clinically significant change in angioedema quality of life score. The most common side effect was gastrointestinal symptoms. In conclusion, preclinical and clinical data indicated that Berotralstat is an effective treatment for long-term prophylactic treatment by suppressing the onset of acute attack in HAE patient and is considered to be a useful treatment option for patients.

Novel pyrazolo[1,5-a]pyrimidines as c-Src kinase inhibitors that reduce IKr channel blockade.

To improve the in vitro potency of the c-Src inhibitor 1a and to address its hERG liability, a structure-activity study was carried out, focusing on two regions of the lead compound. The blockade of the delayed cardiac current rectifier K(+) (I(Kr)) channel was overcome by replacing the ethylenediamino group with an amino alcohol group at the 7-position. In addition, modifying the substituents at the 5-position and the side chain groups on the amino alcohols at the 7-position enhanced the intracellular c-Src inhibitory activity and increased central nervous system (CNS) penetration. In the present study, 6l exhibited significant in vivo efficacy in a middle cerebral artery (MCA) occlusion model in rats.

The importance of CH/pi hydrogen bonds in rational drug design: An ab initio fragment molecular orbital study to leukocyte-specific protein tyrosine (LCK) kinase.

The interaction energy was calculated, by the ab initio FMO method, for complexes between LCK protein and four inhibitors (staurosporine, BMS compound 2, and our compounds 3 and 4). In every case a number of CH/pi hydrogen bonds have been disclosed in the so-called adenine pocket. In complexes of 2, 3, and 4, CH/pi and NH/pi hydrogen bonds have been observed in another pocket. In view of the above results, the aniline ring of 3 was replaced by 2,6-dimethyl aniline to increase the potency for LCK kinase. A 10-fold increase in the potency has been achieved for 4 over 3. We suggest that the concept of weak hydrogen bonds is useful in the rational design of drugs.

The relationship of Prevotella intermedia, Prevotella nigrescens and Prevotella melaninogenica in the supragingival plaque of children, caries and oral malodor.

PURPOSE: A relationship between the distribution of periodontal bacteria species and malodor in children has not been sufficiently investigated. The present study was undertaken to determine the presence of 3 periodontopathic bacteria (Prevotella spp. P. intermedia, P. nigrescens, P. melaninogenica) in the supragingival plaques of 3 to 16-year-old children with different oral health conditions and oral malodor. METHODS: The number of decayed and filled primary teeth (df) and Decayed, Missing and Filled permanent teeth (DMF), Papillary Marginal and Attached gingivitis (PMA) index, Oral Hygiene Index (OHI), and oral malodor of each subject were determined prior to the collection of supragingival plaques. Three periodontopathic bacteria (P. intermedia, P. nigrescens, P. melaninogenica) in supragingival plaques were detected by using an immunoslot blot assay with monoclonal antibodies specific for each microorganism. FINDINGS: The frequencies of periodontopathic bacteria in children with and without caries were not significantly different from each other. Positivity for P. intermedia, but not for P. nigrescens or P. melaninogenica was correlated with oral malodor. Oral malodor was also correlated with the debris index, a component of OHI. The group with the higher OHI showed a higher prevalence of periodontopathic bacteria. For the 3 periodontopathic bacteria in the subjects tested, plaques positive for any of them were not age related. However the frequencies of all 3 periodontopathic bacteria were the highest in the 3-6-year olds. CONCLUSION: The supragingival plaques in children can harbor 3 species of periodontopathic bacteria, P. intermedia, P. nigrescens, and P. melaninogenica.

Morphological and functional changes in cell junctions during secretory stimulation in the perfused rat submandibular gland.

To examine the influence of cholinergic and beta-adrenergic agents on paracellular transport, we applied confocal microscopy and freeze-fracture to the isolated, perfused submandibular gland of the rat. By confocal microscopy, perfusion of lucifer yellow through an arterial catheter, revealed a bright fluorescence in the basolateral spaces of acini, but not in the intercellular canaliculi. However, addition of isoproterenol on carbachol stimulation, induced lucifer yellow fluorescence in intercellular canaliculi. This finding indicates that isoproterenol is capable of opening the paracellular route. The tight junction strands surrounding intercellular canaliculi were visualized using freeze replicas. Fixation was carried out both by vascular perfusion with Karnovsky's solution and by metal contact rapid freezing with liquid helium. In the chemically-fixed specimens, the strand particles of tight junctions formed 2-5 lines at the P-face along most of the apical portion at rest. With carbachol/isoproterenol stimulation, the strand particles rearranged with free ends and terminal loops. In the rapidly frozen specimens, the strand particles were arranged more irregularly even in the resting state. The meshwork of strands became more disheveled and interrupted during carbachol/ isoproterenol stimulation. The present findings led us to conclude that: 1) the beta-adrenergic agent, isoproterenol, can open the paracellular transport. 2) in the rapidly frozen specimen, the tight junction strand particles are arranged roughly and become disheveled and interrupted during stimulation by carbachol/isoproterenol. These findings may be related to rearrangement of subcellular structures, especially of the actin filament network.

Synthesis and c-Src inhibitory activity of imidazo[1,5-a]pyrazine derivatives as an agent for treatment of acute ischemic stroke.

We synthesized and evaluated a series of C-5 substituted imidazo[1,5-a]pyrazine derivatives to identify potent c-Src inhibitors as potential therapeutic agents for acute ischemic stroke. Among these compounds, compound 14c.HCl demonstrated remarkable central nervous system (CNS) penetration and significant neuroprotective efficacy in vivo in rat models.

Discovery of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives as c-Src kinase inhibitors for the treatment of acute ischemic stroke.

We synthesized a series of novel 2-anilinopyrazolo[1,5-a]pyrimidine derivatives and evaluated their ability to inhibit c-Src kinase; 7-(2-amino-2-methylpropylamino)-5-cyclopropyl-2-(3,5-dimethoxyphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide 7o and 7-(2-amino-2-methylpropylamino)-2-(3,5-dimethoxyphenylamino)-5-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide 7f showed potent inhibitory activity. Compound 7f inhibited c-Src selectively and exhibited satisfactory central nervous system (CNS) penetration. Furthermore, 7f.HCl reduced the infarct volume in vivo in a rat middle cerebral artery (MCA) occlusion model when administrated intraperitoneally.

Serine/threonine protein kinase SpkA in Synechocystis sp. strain PCC 6803 is a regulator of expression of three putative pilA operons, formation of thick pili, and cell motility.

Previous studies showed that a Ser/Thr protein kinase, SpkA, in Synechocystis sp. strain PCC 6803 is involved in cell motility. The present study, in which DNA microarray analysis and electron microscopy were used, demonstrated that SpkA regulates the expression of putative pilA9-pilA10-pilA11-slr2018, pilA5-pilA6, and pilA1-pilA2 operons and is essential for the formation of thick pili.

Polyphenolic glycosides and oligosaccharide multiesters from the roots of Polygala dalmaisiana.

Four new polyphenolic glycosides, dalmaisiones A-D (1-4), 16 new oligosaccharide multiesters, dalmaisioses A-P (5, 7-21), and one known tetrasaccharide multiester, reiniose G (6), were isolated from the roots of Polygala dalmaisiana. The structures of the new compounds were elucidated on the basis of chemical and spectroscopic evidence.

A study of lipid secretion from the lichen symbionts, ascomycetous fungus Myelochroa leucotyliza and green alga Trebouxia sp.

Lichens are symbionts of fungi and algae. Although wild fungi secrete lipids to form crystals, those grown in culture either alone or with algae do not secrete enough lipids to be crystallized. To investigate the mode of lipid secretion, we stimulated fungi cultured alone to form crystals. (1) The fungi had serpentine invaginations on the P-faces. These were formed as a consequence of secretory granule exocytosis. (2) Fungi cultured alone in normally used medium had on their P-faces intramembrane particle-cleared parts that also showed a serpentine configuration. (3) After the medium was fortified by further addition of glucose, the fungi cultured alone produced multiple lipid bodies and secretory granules, though no crystals were formed. (4) After the addition of filtered algal culture medium into the fungal culture medium that had been fortified, the fungi grown under this condition showed extracellular crystals. As fungi also showed extensive exocytotic activity, protein secretion seemed essential prior to lipid secretion. The place with no intramembrane particles was postulated to be the site through which lipids penetrate to the outside. (5) As we had identified crystals as atranorin, we made atranorin-containing liposome. The liposome released atranorin to the aqueous phase by the addition of albumin or albumin-like proteins.

Identification of crystalline material found in the thallus of the lichen, Myelochroa leucotyliza.

Lichens are symbiotic associations of fungi and algae (or cyanobacteria). Fungal cells produce large amounts of lipid, assisted by algae, and secrete them out of the cells. Some of the secreted lipids crystallize in the thallus of the lichen. The crystalline materials sometimes occupy 30% of total dry weight of the thallus. This unusual amount of lipid crystal led to our interest in investigating the mechanism of lipid secretion. To begin the cell biological study of lipid secretion and to know the significance of the existence of such crystals in the thallus, it is essential to identify the crystal. The lipid crystal extracted from the thallus of a lichen, Myelochroa leucotyliza, was studied by EM observations, TLC analysis, and EM and X-ray diffraction methods. Atranorin is the predominant component of the crystalline materials in the lipids extracted.