Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1.

Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [14C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC50 values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively) compared with 19 nM for the wild type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC50 of the hydroxyl group methylated dotinurad (F13141) was 165 µM, 8420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC50 value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. SIGNIFICANCE STATEMENT: Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting organic ion transporters (OATs). This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 is found to mediate hydrogen bonding.

Identification of a DBA/2 Mouse Sub-strain as a Model for Pseudoxanthoma Elasticum-Like Tissue Calcification.

Ectopic calcification in the cardiovascular system adversely affects life prognosis. DBA/2 mice experience calcification owing to low expression of Abcc6 as observed in pseudoxanthoma elasticum (PXE) patients; however, little is known about its characteristics as a calcification model. In this study, we explore the suitability of a DBA/2 sub-strain as a PXE-like tissue calcification model, and the effect of a bisphosphonate which prevents calcification of soft tissues in hypercalcemic models was evaluated. The incidence of calcification of the heart was compared among several sub-strains and between both sexes of DBA/2 mice. mRNA expression of calcification-related genes was compared with DBA/2 sub-strains and other mouse strains. In addition, progression of calcification and calciprotein particle formation in serum were examined. Among several sub-strains of DBA/2 mice, male DBA/2CrSlc mice showed the most remarkable cardiac calcification. In DBA/2CrSlc mice, expression of the anti-calcifying genes Abcc6, Enpp1 and Spp1 was lower than that in C57BL/6J, and expression of Enpp1 and Spp1 was lower compared with other sub-strains. Calcification was accompanied by accelerated formation of calciprotein particle, which was prevented by daily treatment with bisphosphonate. A model suitable for ectopic calcification was identified by choosing a sub-strain of DBA/2 mice, in which genetic characteristics would contribute to extended calcification.

Uricosuric Agents Affect Plasma and Kidney Concentration of Adefovir via Inhibition of Oat1 and Mrp2 in Rats.

Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague-Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug-drug interaction risk mediated by OAT1, and also by MRP2.

Visceral Fat Area Measured by Abdominal Bioelectrical Impedance Analysis in School-Aged Japanese Children.

Abdominal bioelectrical impedance analysis (aBIA) has been in use to measure visceral fat area (VFA) in adults. Accurately measuring visceral fat using aBIA in children is challenging. Forty-six school-aged Japanese children aged 6-17 years (25 boys and 21 girls) were included in this study. All were measured, and their VFA obtained using aBIA (VFA-aBIA) and abdominal computed tomography (CT) (VFA-CT) were compared. VFA-aBIA was corrected using the Passing-Bablok method (corrected VFA-aBIA). The relationships between corrected VFA-aBIA and obesity-related clinical factors were analyzed, including non-alcoholic fatty liver disease (NAFLD) and serum leptin and adiponectin levels. Boys had higher VFA-CT than girls (p = 0.042), although no significant differences were found in their waist circumference, waist-to-height ratio, and body mass index. The corrected VFA-aBIA using y = 9.600 + 0.3825x (boys) and y = 7.607 + 0.3661x (girls) correlated with VFA-CT in both boys and girls. The corrected VFA-aBIA in patients with NAFLD was higher than that in those without NAFLD. Serum leptin and adiponectin levels were positively and negatively correlated with corrected VFA-aBIA, respectively. In conclusion, corrected VFA-aBIA was clearly correlated with VFA-CT and was related to NAFLD and serum leptin and adiponectin levels in school-aged Japanese children.

Hypouricemic agents reduce indoxyl sulfate excretion by inhibiting the renal transporters OAT1/3 and ABCG2.

Indoxyl sulfate (IS) accumulates in the body in chronic kidney disease (CKD). In the renal proximal tubules, IS excretion is mediated by OAT1/3 and ABCG2. These transporters are inhibited by some hypouricemic agents; OATs by probenecid and benzbromarone, ABCG2 by febuxostat and benzbromarone. Thus, we evaluated whether hypouricemic agents including dotinurad, a novel selective urate reabsorption inhibitor with minimal effect on OATs or ABCG2, affect IS clearance in rats. Intact and adenine-induced acute renal failure rats were orally administered hypouricemic agents, and both endogenous IS and exogenously administered stable isotope-labeled d4-IS in the plasma and kidney were measured. Our results demonstrated that OATs inhibitors, such as probenecid, suppress IS uptake into the kidney, leading to increased plasma IS concentration, whereas ABCG2 inhibitors, such as febuxostat, cause renal IS accumulation remarkably by suppressing its excretion in intact rats. The effects of these agents were reduced in adenine-induced acute renal failure rats, presumably due to substantial decrease in renal OAT1/3 and ABCG2 expression. Dotinurad did not significantly affected the clearance of IS under both conditions. Therefore, we suggest that hypouricemic agents that do not affect OATs and ABCG2 are effective therapeutic options for the treatment of hyperuricemia complicated by CKD.

Discovery, optimization, and evaluation of non-bile acid FXR/TGR5 dual agonists.

Although several potent bile acid Farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5, GPBAR1) dual agonists such as INT-767 have been reported, no non-bile acid FXR/TGR5 dual agonist has been investigated to date. Therefore, we attempted to discover potent non-bile acid FXR/TGR5 dual agonists and identified some non-bile acid FXR/TGR5 dual agonists, such as isonicotinamide derivatives in vitro assay. Compound 20p was evaluated in C57BL/6J mice, that were administered a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight for one week. Compound 20p dose-dependently induced small heterodimer partner (SHP) mRNA and decreased cytochrome P450 7A1 (CYP7A1) in the liver at 10 and 30 mg/kg, respectively, which were used as FXR agonist markers. Compound 20p significantly increased the plasma levels of GLP-1 as a TGR5 agonist, and a high concentration of GLP-1 lowered blood glucose levels. We confirmed that compound 20p was a non-bile acid FXR/TGR5 dual agonist.

Dotinurad: a novel selective urate reabsorption inhibitor for the treatment of hyperuricemia and gout.

INTRODUCTION: Gout is an inflammatory disease triggered by deposition of urate crystals secondary to longstanding hyperuricemia, and its management implies both the treatment of flares and management of hyperuricemia. Dotinurad is a selective urate reabsorption inhibitor (SURI), potently inhibits urate transporter 1 in the apical surface of renal proximal tubular cells, and has been approved for the treatment of gout and hyperuricemia in Japan. AREAS COVERED: This overview of dotinurad covers nonclinical and clinical pharmacology studies in special populations and its efficacy and safety in Japanese hyperuricemic patients with and without gout. EXPERT OPINION: Dotinurad, as an SURI, is expected to inhibit urate reabsorption more effectively than conventional urate-lowering agents. It is noninferior to benzbromarone or febuxostat in reducing serum urate levels in hyperuricemic patients with or without gout. Its efficacy is not attenuated in patients with mild to moderate renal impairment or with hepatic impairment. At a maintenance dose of 2 or 4 mg once daily, most patients achieved the target serum urate level of ≤6 mg/dL in a long-term study. No findings that raised safety concerns, including liver injury, were identified. Dotinurad is expected to be a new therapeutic option in hyperuricemic patients with and without gout.

Successful de-escalation antibiotic therapy using cephamycins for sepsis caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae bacteremia: A sequential 25-case series.

Carbapenems are frequently used to treat infections caused by extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E), but carbapenem-resistant Enterobacteriaceae bacteria are a clinical concern. Although cephamycins (cefmetazole; CMZ) have been shown to be effective against mild cases of ESBL-E infection, data on their use for severe ESBL-E infections with sepsis or septic shock remain scarce. Herein, we discuss a de-escalation therapy to CMZ that could be used after empiric antibiotic therapy in ICU patients with sepsis or septic shock caused by ESBL-E bacteremia. A sequence of 25 cases diagnosed with sepsis or septic shock caused by ESBL-E bacteria was evaluated. The attending infectious disease specialist physicians selected the antibiotics and decided the de-escalation timing. The median SOFA (Sequential Organ Failure Assessment) and APACHE II (Acute Physiology and Chronic Health Evaluation II) severity scores were 8 and 30; the rate of septic shock was 60%. Infections originated most frequently with urinary tract infection (UTI) (56%) and Escherichia coli (85%). Eleven patients were de-escalated to CMZ after vital signs were stable, and all survived. No patients died of UTI regardless of with or without de-escalation. The median timing of de-escalation antibiotic therapy after admission was 4 days (range, 3-6 days). At the time of de-escalation, the median SOFA score fell from 8 to 5, the median APACHE II score from 28 to 22, and the rate of septic shock from 55% to 0%. We conclude that for sepsis in UTI caused by ESBL-E bacteremia, de-escalation therapy from broad-spectrum antibiotics to CMZ is a potential treatment option when vital signs are stable.

[Pharmacological properties and clinical efficacy of dotinurad (URECE® tablets), a novel hypouricemic agent].

In Jan 2020, dotinurad (URECE® tablets) was approved for gout and hyperuricemia therapy in Japan. We developed a novel hypouricemic agent because benzbromarone, a commercially available uricosuric agent, has several problems, such as drug-induced liver injury or drug-drug interaction caused by CYP2C9 inhibition. In transporter-overexpressing cells, dotinurad potently inhibited URAT1 which is localized in the renal proximal tubules and functions as a urate reabsorption. On the contrary, dotinurad hardly inhibited urate secretion transporters, ABCG2 or OAT1/3. In Cebus monkeys, dotinurad dose-dependently decreased plasma urate levels at low doses compared with benzbromarone. Inhibitory effect of dotinurad on mitochondria was weaker than that of benzbromarone and there was no observation suggesting a risk of drug-induced liver injury taking into consideration the clinical dose or exposure. Dotinurad weakly inhibited CYPs and further analysis indicated there was no drug-drug interaction risk in the clinical dose. In clinical pharmacology studies, there was no difference among sex and age. Furthermore, dosage and administration are equal even in hepatic impairment patients (mild to severe) and renal impairment patients (mild to moderate). In confirmatory phase II and long-term studies, dotinurad decreased serum urate levels at low doses and almost patients using maintenance dose (2 or 4 mg) achieved a serum urate level ≤ 6.0 mg/dL. Moreover, there was no finding to raise safety concern including liver injury. In conclusion, dotinurad, a selective urate reabsorption inhibitor (SURI) could be a therapeutic option because of its more effective hypouricemic action at low doses than those of commercially available uricosuric agents.

Pharmacological Evaluation of Dotinurad, a Selective Urate Reabsorption Inhibitor.

The effect of dotinurad [(3,5-dichloro-4-hydroxyphenyl)(1,1-dioxo-1,2-dihydro-3H-1λ 6-1,3-benzothiazol-3-yl)methanone] was compared with that of commercially available uricosuric agents-namely, benzbromarone, lesinurad, and probenecid. Its effect on urate secretion transporters was evaluated using probe substrates for respective transporters. Dotinurad, benzbromarone, lesinurad, and probenecid inhibited urate transporter 1 (URAT1) with IC50 values of 0.0372, 0.190, 30.0, and 165 µM, respectively. Dotinurad weakly inhibited ATP-binding cassette subfamily G member 2 (ABCG2), organic anion transporter 1 (OAT1), and OAT3, with IC50 values of 4.16, 4.08, and 1.32 µM, respectively, indicating higher selectivity for URAT1. The hypouricemic effects of dotinurad and benzbromarone were evaluated in Cebus monkeys. Dotinurad, at doses of 1-30 mg/kg, concomitantly decreased plasma urate levels and increased fractional excretion of urate (FEUA) in a dose-dependent manner. On the contrary, benzbromarone, at a dose of 30 mg/kg, showed a modest effect on plasma urate levels. The inhibitory effect of dotinurad on urate secretion transporters was evaluated in Sprague-Dawley rats, with sulfasalazine and adefovir as probe substrates of ABCG2 and OAT1, respectively. Drugs, including febuxostat as a reference ABCG2 inhibitor, were administered orally before sulfasalazine or adefovir administration. Dotinurad had no effect on urate secretion transporters in vivo, whereas benzbromarone, lesinurad, probenecid, and febuxostat increased the plasma concentrations of probe substrates. These results suggested dotinurad is characterized as a selective urate reabsorption inhibitor (SURI), which is defined as a potent URAT1 inhibitor with minimal effect on urate secretion transporters, including ABCG2 and OAT1/3, because of its high efficacy in decreasing plasma urate levels compared with that of other uricosuric agents. SIGNIFICANCE STATEMENT: Our study on the inhibitory effects on urate transport showed that dotinurad had higher selectivity for urate transporter 1 (URAT1) versus ATP-binding cassette subfamily G member 2 (ABCG2) and organic anion transporter (OAT) 1/3 compared to other uricosuric agents. In Cebus monkeys, dotinurad decreased plasma urate levels and increased fractional excretion of urate in a dose-dependent manner. To determine the inhibitory effect of dotinurad on urate secretion transporters, we studied the movement of substrates of ABCG2 and OAT1 in rats. Dotinurad had no effect on these transporters, whereas the other uricosuric agents increased the plasma concentrations of the substrates. These results suggested dotinurad as a potent and selective urate reabsorption inhibitor is characterized by increased efficacy with decreasing plasma urate levels.

Seasonal changes in sleep duration and sleep problems: A prospective study in Japanese community residents.

BACKGROUND: A scientific understanding of the effects of seasonal changes on sleep duration and sleep problems such as insomnia and hypersomnia has yet to be elucidated; however, such an understanding could aid the establishment of an optimal sleep hygiene program to treat such problems. METHODS: We investigated the effects of seasonal changes on sleep duration and sleep problems in Japanese community residents. Data on 1,388 individuals aged 15-89 years who participated in the Survey of Seasonal Variations in Food Intakes conducted by the National Institute of Health and Nutrition of Japan (2004-2007) were analyzed. Participants completed a questionnaire including items on sleep duration and sleep problems (difficulty initiating sleep [DIS], difficulty maintaining sleep [DMS]/early morning awakening [EMA], and excessive daytime sleepiness [EDS]). Data were prospectively collected at four time points (spring, summer, fall, and winter). RESULTS: Seasonal changes in sleep duration were found, with the longest in winter and the shortest in summer (winter-summer difference: 0.19 h). The seasonality of sleep duration was influenced by age, sex, and residential area. In terms of age, seasonal changes in sleep duration were found in the middle and old age groups, but not in the young age group. Seasonal changes in the frequencies of sleep problems were found for some items in the young age group (DMS/EMA and EDS) and middle age group (DIS and DMS/EMA); however, no such changes were observed in the old age group. CONCLUSION: Seasonal effects on sleep and sleep problems were found in Japanese community residents, but these varied between age groups. Furthermore, seasonal changes in sleep duration were influenced by sex and residential area.

Enhancement of pharmacological effects of uricosuric agents by concomitant treatment with pyrazinamide in rats.

Our goal was to establish a model for the evaluation of the effects of uricosuric agents and to clarify the underlying mechanism(s). The effects of a uricosuric agent co-treated with pyrazinamide, an anti-tubercular agent, on urate handling were examined in rats. Furthermore, the effects of uricosuric agents on urate uptake were evaluated using the vesicles of rat renal brush-border membrane. Treatment with probenecid, at a dose of 100 mg/kg, significantly increased the urinary urate to creatinine ratio (UUA/UCRE) in pyrazinamide-treated rats although the same treatment did not produce any uricosuric effects in intact rats. In this model, the urinary excretion of pyrazinecarboxylic acid (PZA), an active metabolite of pyrazinamide, was decreased by probenecid and indicated an inverse correlation between urinary excretion of urate and PZA. Furthermore, in the examination using FYU-981, a potent uricosuric agent, a more than 10-fold leftward shift of the dose-response relationship of the uricosuric effect was observed in pyrazinamide-treated rats when compared with intact rats. In the in vitro study, the treatment of the vesicles of rat renal brush-border membrane with PZA produced an increased urate uptake, which was inhibited by uricosuric agents. The pyrazinamide-treated model used in the present study seems to be valuable for the evaluation of uricosurics because of its higher sensitivity to these drugs when compared to intact rats, and this is probably due to the enhanced urate reabsorption accompanied with trans-stimulated PZA transport at the renal brush-border membrane.

Uricosuric agents decrease the plasma urate level in rats by concomitant treatment with topiroxostat, a novel xanthine oxidoreductase inhibitor.

OBJECTIVES: The aim of this study was to establish the rat model for evaluating hypouricemic effects by some uricosuric agents. METHODS: Rats were made hyperuricemic by subcutaneous administration of potassium oxonate, a uricase inhibitor, or made hypouricemic by oral administration of topiroxostat, a xanthine oxidoreductase inhibitor. Furthermore, rats were co-treated with topiroxostat and inosine, a urate precursor. In each condition, hypouricemic effects by uricosuric agents were examined. KEY FINDINGS: In potassium oxonate-treated rats, treatment with uricosuric agents such as FYU-981, F12859 and probenecid showed no hypouricemic effect. On the other hand, in topiroxostat-treated rats, uricosuric agents remarkably lowered plasma urate level compared with topiroxostat treatment alone, with a dose dependency of 30 and 100 mg/kg for FYU-981 and F12859 each. The decrease in the plasma urate level observed in the topiroxostat-treated rats disappeared by further co-treatment with inosine. CONCLUSIONS: Effects of uricosuric agents on the plasma urate level in rats were sensitive to the rate of urate formation. Induction of slower urate formation by topiroxostat provides valuable model for evaluation of hypouricemic effects by uricosuric agents in rats.

Establishment of simultaneous treatment model with citrate for preventing nephropathy induced by FYX-051, a xanthine oxidoreductase inhibitor, in rats.

As a precedent study for elucidating the mechanism of possible urinary bladder carcinogenesis due to xanthine crystals induced by FYX-051, a xanthine oxidoreductase inhibitor, we have determined the experimental conditions suitable for the 52-week simultaneous treatment with citrate in F344 rats. Simultaneous treatment with citrate and FYX-051 produced both increased urinary citrate excretion and suppression of urinary xanthine deposition at around 4 hours after a single dosing, but these effects disappeared 2 hours later, indicating a lack of the durability of citrate effects. Next, we carried out a 7-day simultaneous treatment study by two daily treatments, that is, FYX-051 (6 mg/kg) and citrate (2,000 mg/kg), followed by citrate-alone treatment, under the conditions of selected dosing intervals, the second dose of citrate, and dosing volume. As a result, the dosing interval of citrate was found to be optimal at 4 hours, but not at 3 or 5 hours, because this treatment completely inhibited intrarenal xanthine deposition. The dose of citrate for the second treatment and the dosing volume were found to be sufficient at 1,500 mg/kg and 10 mL/kg, respectively. Subsequently, a 4-week study by simultaneous treatment at 3 mg/kg of FYX-051 and citrate (2,000 mg/kg) + citrate (1,500 mg/kg), under the improved conditions, revealed that renal lesions could be drastically inhibited. Thus, the present study demonstrated that the interval of two citrate treatments is pivotal and indicated that the improved model would be useful for the mechanistic study of FYX-051-induced urinary bladder carcinogenesis because of an easier treatment method than our previous model.

Alanine scanning analyses of the three major loops in domain II of Bacillus thuringiensis mosquitocidal toxin Cry4Aa.

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

Elevated production of Legionella-specific immunoglobulin A in A/J mice is accompanied by T-helper 1-type polarization.

Legionella pneumophila (Lpn) is a Gram-negative bacterium and an intracellular parasite that causes Legionnaires' disease. Secretion of immunoglobulin A (IgA) against Lpn on the mucosal surface of the upper respiratory tract may be important as a self-defense mechanism. A/J mice have been demonstrated to be permissive as to Lpn replication in macrophages due to a natural mutation in neuronal apoptosis inhibitory protein 5. We compared A/J and BALB/c mice as to IgA production after repeated intranasal immunization using a heat-killed Lpn in the presence of cholera toxin as a mucosal adjuvant. A/J mice secreted more Lpn-specific IgA in nasal washes than BALB/c mice. The Lpn-specific serum IgA level was also higher in A/J than BALB/c mice. Because both BALB/c and A/J mice are known to exhibit T-helper 2 (Th2)-biased immune responses, we examined whether the Lpn-specific IgA production is related to the stronger Th2 bias. There was no difference in IgG1 (Th2-controlled) while A/J mice produced more IgG2a (Th1-controlled), suggesting that the elevated IgA response was rather correlated with Th1-controlled isotype switching. Our results also suggest that A/J mice will be useful hosts for Lpn-specific IgA production such as for the preparation of IgA monoclonal antibodies.