Protein engineering of lipase A from Candida antarctica to improve esterification of tertiary alcohols.

Chiral tertiary alcohols are important organic compounds in science as well as in industry. However, their preparation in enantiomerically pure form is still a challenge due to their complex structure and steric hindrances compared with primary and secondary alcohols, so kinetic resolution could be an attractive approach.  Lipase A from Candida antarctica (CAL-A) has been shown to catalyze the enantioselective esterification of various tertiary alcohols with excellent enantioselectivity but low activity. Here we report a mutagenesis study by rational design to improve CAL-A activity against tertiary alcohols. Single mutants of CAL-A were selected, expressed, immobilized and screened for esterification of the tertiary alcohol 1,2,3,4-tetrahydronaphthalene-1-ol. A double mutant V278S+S429G showed a 1.5-fold higher reaction rate than that of the wild type CAL-A, while maintaining excellent enantioselectivity.

Strategies to Design Chemocatalytic Racemization of Tertiary Alcohols: State of the Art & Utilization for Dynamic Kinetic Resolution.

The synthesis of enantiomerically pure tertiary alcohols is an important issue in organic synthesis of a range of pharmaceuticals including molecules such as the anti-HIV drug Efavirenz. A conceptually elegant approach to such enantiomers is the dynamic kinetic resolution of racemic tertiary alcohols, which, however, requires efficient racemization strategies. The racemization of tertiary alcohols is particularly challenging due to various side reactions that can occur because of their high tendency for elimination reactions. In the last few years, several complementary catalytic concepts for racemization of tertiary alcohols have been developed, characterized by efficient racemization and suppression of unwanted side-reactions. Besides resins bearing sulfonic acid moieties and a combination of boronic acid and oxalic acid as heterogeneous and homogeneous Brønsted-acids, respectively, immobilized oxovanadium and piperidine turned out to be useful catalysts. The latter two catalysts, which have already been applied to different types of substrates, also have proven good compatibility with lipase, thus leading to the first two examples of chemoenzymatic dynamic kinetic resolution of tertiary alcohols. In this review, the difficulties in racemizing tertiary alcohols are specifically described, and the recently developed complementary concepts to overcome these hurdles are summarized.

Topical therapy and skin care for transplant-associated atopic dermatitis in children and adolescents.

BACKGROUND: New-onset allergic diseases, such as food allergy or atopic dermatitis, can develop after allogeneic transplantation. There are limited reports of new-onset atopic dermatitis after allogeneic hematopoietic stem cell transplantation in children and adolescents, and its treatment is yet to be established. The pathogenesis may differ from typical atopic dermatitis in terms of alloimmunity including graft-versus-host disease. METHODS: We present five children and adolescents with new-onset atopic dermatitis after allogeneic hematopoietic stem cell transplantation. The characteristics and clinical profiles of skin treatment after hematopoietic stem cell transplantation are summarized. RESULTS: Graft-versus-host disease prophylaxis included systemic tacrolimus for all patients. After hematopoietic stem cell transplantation, all patients achieved complete donor chimerism of the bone marrow and had acute graft-versus-host disease of the skin. After engraftment, all patients had skin lesions that met the international consensus diagnostic criteria for atopic dermatitis. None of the patients met the diagnostic criteria for chronic graft-versus-host disease. Topical therapy and skin care based on atopic dermatitis guidelines improved skin condition and atopic dermatitis severity scores in all patients. In addition, type 2 inflammatory markers improved accordingly. CONCLUSION: Topical therapy and skin care may be effective for transplant-related atopic dermatitis after hematopoietic stem cell transplantation. When extensive dermatitis is observed after hematopoietic stem cell transplantation, this treatment may avoid excessive immunosuppressive therapy if it meets the diagnostic criteria for atopic dermatitis.

[A SURVEY ON MEDICAL CARE FOR ALLERGIC DISEASES IN MIYAGI].

OBJECTIVE: In Miyagi, the number of allergy specialists per population is higher at Sendai city compared to the other areas (non-Sendai areas). Therefore, the healthcare delivery for allergic diseases are unevenly distributed. In the current study, we investigated differences of medical care for allergic diseases between Sendai city and non-Sendai areas. METHODS: We conducted a web-based questionnaire survey to all of hospitals and clinics in the prefecture. The questionnaire responses were analyzed and compared between the Sendai city and non-Sendai areas. RESULTS: Responses to the questionnaire were obtained from 175 hospitals and clinics, including 72 internal physicians, 34 pediatricians, 17 dermatologists, 15 otorhinolaryngologists, 12 ophthalmologists and 25 others. More clinicians in non-Sendai areas felt the difficulty in treating asthma and chronic urticaria than those in Sendai city. Fewer institutions prescribed biologics for severe allergic diseases in non-Sendai areas than in Sendai city, which might be due to the lack of knowledge on the biologic agents. On the other hand, referring patients with anaphylaxis to specialized hospitals tended to be more difficult in Sendai city compared to in non-Sendai areas. Additionally, the regional medical liaison system is needed to refer patients with severe allergic diseases to advanced medical institutions. CONCLUSION: There are unique problems about allergy care in Miyagi.

[CLINICAL MANIFESTATIONS AND SAFETY OF AN ORAL FOOD CHALLENGE USING BOILED EGG YOLK].

BACKGROUND: Allergenicity to heated egg yolks is known to be low in hen's egg allergy. However, there are few reports concerning the safety of an oral food challenge (OFC) with boiled egg yolks. This study aimed to determine the safety of a boiled egg yolk OFC and the clinical characteristics of patients who were OFC-positive. METHODS: We retrospectively examined the data of patients who underwent an OFC with one boiled egg yolk at Miyagi Children's Hospital between January 2013 and December 2020. Patients were included if they had a history of immediate symptoms due to hen's egg or positive specific IgE levels to egg yolk or egg white. RESULTS: Among 600 patients, 15.0% were positive for OFC, only one patient required an intramuscular adrenaline injection, and 70% of OFC-positive patients had gastrointestinal symptoms. CONCLUSION: Boiled egg yolk OFC had a low symptom induction rate; however, when symptoms were induced, gastrointestinal symptoms were most commonly observed. Given the low risk of developing severe symptoms, we conclude that the likelihood of safely performing an OFC is high.

Enantiodivergent Chemoenzymatic Dynamic Kinetic Resolution: Conversion of Racemic Propargyl Alcohols into Both Enantiomers.

Natural lipases typically recognize enantiomers of alcohols based on the size differences of substituents near the carbinol moiety and selectively react with the R enantiomers of secondary alcohols. Therefore, lipase-catalyzed dynamic kinetic resolution (DKR) of racemic secondary alcohols produces only R enantiomers. We report herein a method for obtaining S enantiomers by DKR of secondary 3-(trialkylsilyl)propargyl alcohols by using a well-known R-selective Pseudomonas fluorescens lipase in combination with a racemization catalyst VMPS4, in which the silyl group reverses the size relationship of substituents near the carbinol moiety. We have already reported R-selective DKR of the corresponding propargyl alcohols without substituents on the ethynyl terminal carbon, and the presence of an easily removable silyl group has enabled us to produce both enantiomers of propargyl alcohols in high chemical yields and with high enantiomeric excess. In addition, immobilization of the lipase on Celite was found to be important for achieving a high efficiency of the DKR.

Lipase-Catalyzed Kinetic Resolution of C1-Symmetric Heterocyclic Biaryls.

The highly enantioselective lipase-catalyzed kinetic resolution (KR) of racemic C1-symmetric biaryl compounds including heterocyclic moieties, such as carbazole and dibenzofuran, has been achieved for the first time. This enzymatic esterification was accelerated by the addition of disodium carbonate while maintaining its high enantioselectivities, and was particularly effective for biaryls having N-substituted carbazole moieties. Furthermore, mesoporous silica-supported oxovanadium-catalyzed cross-dehydrogenative coupling of 3-hydroxycarbazole and 2-naphthol was followed by the lipase-catalyzed KR in one-pot to synthesize the optically active heterocyclic biaryl compounds with high optical purity.

Differences in allergic symptoms after the consumption of egg yolk and egg white.

Hen's eggs are one of the most common causes of food allergy. Although hen's eggs are known to cause more gastrointestinal symptoms than other foods, it is not known whether there is a difference in organ-specific symptoms between egg yolk (EY) and egg white (EW). The present study aimed to determine whether there are organ-specific differences in the immediate symptoms of EY and EW in patients with hen's egg allergies. We retrospectively investigated the immediate symptoms and treatment contents of those who had a positive result in an oral food challenge (OFC) of boiled whole EY or 10 g of boiled EW in our hospital from January 2013 to July 2019. We compared 80 patients in the EY-OFC-positive group with 106 patients in the EW-OFC-positive group. The EY-OFC-positive group had significantly fewer respiratory symptoms and significantly more gastrointestinal symptoms than the EW-OFC-positive group and had significantly more gastrointestinal symptoms only. In terms of treatment, significantly fewer patients in the EY-OFC-positive group required beta 2-agonist inhalation, and a significantly higher proportion of patients did not require treatment. Compared to EW, EY is more likely to cause gastrointestinal symptoms and less likely to cause respiratory symptoms. It may be necessary to discriminate between EY and EW allergy during diagnosis.

Use of a home-visit nursing service to manage severe atopic dermatitis in a child with difficult family environment.

Maintenance therapy of atopic dermatitis (AD) with frequent exacerbations needs proactive use of topical corticosteroids or topical calcineurin inhibitors. However, it can be difficult for children in an unfavorable family environment. Home-visit nursing service (HVNS) plays an important role in delivering care to people who need medical help at home. We report a boy in a difficult family situation who received HVNS to successfully manage AD.

Home-based, slow up-dosing oral immunotherapy for hen's egg allergy in an adult patient.

Standard therapy for food allergies involves avoiding causative foods until a patient has outgrown their allergies. Oral immunotherapy (OIT) is an optional treatment for children unlikely to outgrow their food allergy. However, information about OIT in adult patients with food allergies is very limited. We present a case of severe hen's egg allergy (HEA) in an adult who tried home-based, slow up-dosing OIT, reported to have been tolerable and effective in children. A 20-year-old woman with HEA experienced repeated anaphylaxis since childhood when she consumed a small quantity of hen's egg, so she completely avoided hen's eggs. She underwent inpatient oral food challenge (OFC) with 10-g boiled egg yolk and presented lip swelling and abdominal pain. OFC with 1-g boiled egg yolk the following day induced no adverse reaction. OIT was initiated using a home-based, slow up-dosing protocol. She consumed 1 g of boiled egg yolk at home every day, increasing this by 5%-10% every 2 weeks. She started 0.5-g boiled egg white after reaching a whole egg yolk. If adverse reactions occurred, the daily dose was decreased. After 59 months, she was able to eat an entire boiled egg. Anaphylaxis occurred 3 times during OIT due to accidental consumptions of egg products or insufficient heating of egg. Home-based, slow up-dosing OIT might be applicable for adults with severe HEA. It should be performed with appropriate equipment and education for patients, in case of emergency.

[CHARACTERISTICS OF THE SYMPTOMS DURING FOOD AND EXERCISE PROVOCATION TESTS IN PATIENTS AFTER ORAL IMMUNOTHERAPY: COMPARISON WITH FOOD-DEPENDENT EXERCISE-INDUCED ANAPHYLAXIS].

OBJECTIVE: We aim to examine the characteristics of the symptoms induced by exercise provocation tests following allergen consumption in patients who have undergone oral immunotherapy (OIT). METHOD: Patients who were positive for exercise provocation tests in Miyagi Children's Hospital from April 2012 to January 2019 were retrospectively analyzed. Patients were classified into food-dependent exerciseinduced anaphylaxis (FDEIA) group and post-OIT-FDEIA group. RESULT: Six patients in the FDEIA group and 19 patients in the post-OIT-FDEIA group were analyzed. There were no significant differences in age, sex, and number of complications between the FDEIA and post-OIT-FDEIA groups, except for level of total serum immunoglobulin E. The median time from the start of exercise to onset of symptoms was 20 min in the FDEIA group and 25 min in the post-OIT-FDEIA group. The rate of adrenaline administration in post-OIT-FDEIA group was significantly lower. The median time from exercise to onset of severe symptoms requiring adrenaline was 32.5 min in the FDEIA group and 25 min in the post-OIT-FDEIA group. CONCLUSION: These data suggest that FDEIA and post-OIT-FDEIA patients can exhibit similar clinical symptoms, and all symptoms occurred in 60 minutes after exercise.

[PROVOCATION TESTS OF FOOD AND EXERCISE AFTER ORAL IMMUNOTHERAPY].

BACKGROUND: Oral immunotherapy (OIT) for food allergy requires exercise restrictions following the regular consumption of allergens to prevent immediate symptoms. We investigated the efficacy and the safety of exercise provocation tests to assess exercise tolerance after consumption of allergens following OIT. METHODS: This study was based on data from patients who underwent exercise provocation tests with consumption of their allergen after OIT in Miyagi Children's Hospital between April 2012 and March 2019. The results of exercise provocation tests and patients' characteristics were retrospectively analyzed. RESULTS: Fifteen out of 91 patients were positive for exercise provocation tests. The positive rates were 12% (4/33) with egg, 15% (5/34) with cow's milk, and 25% (6/24) with wheat. Intramuscular adrenaline injections were administered to two patients with allergies to egg, one with cow's milk, and one with wheat. CONCLUSION: Following OIT, the assessment using exercise provocation tests should be performed prior to termination of exercise restriction because some patients remain intolerant to exercise.

[SAFETY OF A THREE-LEVEL STEPWISE ORAL FOOD CHALLENGE FOR PEANUT ALLERGY].

BACKGROUND: The present study aimed to assess the safety of a three-level stepwise oral food challenge (OFC) in patients with peanut allergy. METHODS: This retrospective analysis reviewed laboratory test results and food allergy histories (obtained from medical records) of participants who underwent OFC using peanut butter for the first time at Miyagi Children's Hospital between January 2015 and December 2017. Total food dose was classified as follows: step 1, 0.1 g peanut butter; step 2, 0.5g peanut butter; and step 3, 3g peanut butter. RESULTS: A total of 114 participants were included. OFC-positive rates were 52% (step 1), 35% (step 2), and 20% (step 3). Of the 41 OFC-positive participants, 1 required an intramuscular adrenaline injection during step 3. Peanut allergy history rates and peanut- and Ara h 2-specific immunoglobulin E (sIgE) titers were significantly higher during the low-dose step. CONCLUSION: The three-level stepwise OFC with peanut butter is safe for patients with peanut allergy, as indicated by peanut- and Ara h 2-sIgE titers.

Hyperresponsiveness to Boiled Egg Yolk in Early Life Leads to Prolonged Egg Allergy.

Hen's egg is the most common allergen in IgE-mediated food allergy among children in Japan. Although the majority of patients with egg allergy can eat heated egg yolk safely because of its low allergenicity, severely allergic patients show an immediate-type reaction to heated egg yolk. We hypothesized that patients with hyperresponsiveness to boiled egg yolk may have difficulty in acquiring tolerance to egg. The purpose of this study was to examine the prognosis of patients with hyperresponsiveness to boiled egg yolk. Data from 121 patients with egg allergy who underwent oral food challenge (OFC) with boiled egg yolk between January 2012 and December 2013 were analyzed retrospectively. The proportion of patients who could consume heated whole egg 3 years after OFC was 15.4% in the OFC-positive group and 75.8% in the OFC-negative group. Hyperresponsiveness to boiled egg yolk in early life might lead to prolonged egg allergy in children. This finding might aid in the selection of an appropriate population requiring practical immunotherapy.

Selective expansion of donor-derived regulatory T cells after allogeneic bone marrow transplantation in a patient with IPEX syndrome.

IPEX syndrome is a rare and fatal disorder caused by absence of regulatory T cells (Tregs) due to congenital mutations in the Forkhead box protein 3 gene. Here, we report a patient with IPEX syndrome treated with RIC followed by allogeneic BMT from an HLA-matched sibling donor. We could achieve engraftment and regimen-related toxicity was well tolerated. Although the patient was in mixed chimera and the ratio of donor cells in whole peripheral blood remained relatively low, selective and sustained expansion of Tregs determined as CD4+CD25+Foxp3+ cells was observed. Improvement in clinical symptoms was correlated with expansion of donor-derived Tregs and disappearance of anti-villin autoantibody, which was involved in the pathogenesis of gastrointestinal symptoms in IPEX syndrome. This clinical observation suggests that donor-derived Tregs have selective growth advantage in patients with IPEX syndrome even in mixed chimera after allogeneic BMT and contribute to the control of clinical symptoms caused by the defect of Tregs.