HIF-2α Inhibits Ameloblast Differentiation via Hey2 in Tooth Development.

Enamel is the highly mineralized outer layer of teeth; the cells responsible for enamel formation are ameloblasts. Local hypoxia and hypoxia inducible factor (HIF) in embryonic tissues are important to promote normal organogenesis. However, hypoxic state in tooth germs and the roles of HIF in ameloblast differentiation have not been understood. The aim of this study is to clarify the role of HIF in ameloblast differentiation during tooth germ development. We found that tooth germs were under hypoxia and HIF-1α and HIF-2α were expressed in tooth germs in embryonic mice. Then, we used HIF inhibitors to evaluate the function of HIF during tooth germ development. The HIF-2α inhibitor significantly decreased the size of tooth germs in organ culture, while the HIF-1α inhibitor did not apparently affect the size of tooth germs. The HIF-2α inhibitor enhanced the expression of amelogenin, a marker of ameloblast differentiation, in the tooth germs in organ culture and rat dental epithelial SF2 cells. Moreover, we found that the HIF-2α inhibitor-stimulating amelogenin expression was regulated by hes-related family basic helix-loop-helix transcription factor with YRPW motif 2(Hey2) in SF2 cells. These findings suggest that the HIF-2α-Hey2 axis plays an important role in ameloblast differentiation during tooth germ development.

Integration of Single-Cell RNA- and CAGE-seq Reveals Tooth-Enriched Genes.

Organ development is dictated by the regulation of genes preferentially expressed in tissues or cell types. Gene expression profiling and identification of specific genes in organs can provide insights into organogenesis. Therefore, genome-wide analysis is a powerful tool for clarifying the mechanisms of development during organogenesis as well as tooth development. Single-cell RNA sequencing (scRNA-seq) is a suitable tool for unraveling the gene expression profile of dental cells. Using scRNA-seq, we can obtain a large pool of information on gene expression; however, identification of functional genes, which are key molecules for tooth development, via this approach remains challenging. In the present study, we performed cap analysis of gene expression sequence (CAGE-seq) using mouse tooth germ to identify the genes preferentially expressed in teeth. The CAGE-seq counts short reads at the 5'-end of transcripts; therefore, this method can quantify the amount of transcripts without bias related to the transcript length. We hypothesized that this CAGE data set would be of great help for further understanding a gene expression profile through scRNA-seq. We aimed to identify the important genes involved in tooth development via bioinformatics analyses, using a combination of scRNA-seq and CAGE-seq. We obtained the scRNA-seq data set of 12,212 cells from postnatal day 1 mouse molars and the CAGE-seq data set from postnatal day 1 molars. scRNA-seq analysis revealed the spatiotemporal expression of cell type-specific genes, and CAGE-seq helped determine whether these genes are preferentially expressed in tooth or ubiquitously. Furthermore, we identified candidate genes as novel tooth-enriched and dental cell type-specific markers. Our results show that the integration of scRNA-seq and CAGE-seq highlights the genes important for tooth development among numerous gene expression profiles. These findings should contribute to resolving the mechanism of tooth development and establishing the basis for tooth regeneration in the future.

Phosphate-sensing and regulatory mechanism of FGF23 production.

BACKGROUND: Inorganic phosphate (Pi) is an essential mineral for human. Hypophosphatemia and hyperphosphatemia cause rickets/osteomalacia and ectopic calcification, respectively, indicating that serum Pi level needs to be regulated. Fibroblast growth factor (FGF) 23 is a principal hormone to regulate serum Pi level. FGF23 is produced by the bone, especially by the osteoblasts and osteocytes, and works by binding to FGF receptor (FGFR) 1c and α-Klotho complex in the kidney. FGF23 reduces serum Pi level by inhibiting both renal phosphate reabsorption and intestinal phosphate absorption via reduction of serum 1,25-dihydroxyvitamin D level. It has been unclear how the bone senses changes of serum Pi level and how the bone regulates the production of FGF23. RECENT FINDINGS: Our recent results indicate that the post-translational modification of FGF23 protein through a gene product of GALNT3 is the main regulatory mechanism of enhanced FGF23 production by high dietary Pi. Furthermore, high extracellular Pi directly activates FGFR1 and its downstream intracellular signaling pathway regulates the expression level of GALNT3. CONCLUSIONS: We propose that FGFR1 works as a Pi-sensing receptor in the regulation of FGF23 production and serum Pi level. There is a negative feedback system, which is a basic mechanism of endocrine regulation, in the regulation of serum Pi involving FGFR1, and FGF23. These findings may lead to the development of new therapeutic methods to treat diseases caused by abnormal Pi level.

Spinal cord involvement by atrophy and associations with disability are different between multiple sclerosis and neuromyelitis optica spectrum disorder.

BACKGROUND AND PURPOSE: The cervical and thoracic cross-sectional spinal cord area (CS-SCA) in multiple sclerosis (MS) correlates with disability, whilst such a correlation remains to be established in neuromyelitis optica spectrum disorder (NMOSD). Our aim was to clarify differences between MS and NMOSD in spinal cord segments where CS-SCA is associated with disability. METHODS: The CS-SCA at C2/C3, C3/C4, T8/T9 and T9/T10 vertebral disc levels was measured in 140 MS patients (111 with relapsing-remitting MS and 29 with progressive MS) and 42 NMOSD patients with anti-aquaporin-4 immunoglobulin G. Disability was evaluated by Expanded Disability Status Scale (EDSS) scores. Multivariate associations between CS-SCA and disability were assessed by stepwise forward multiple linear regression. RESULTS: Thoracic CS-SCA was significantly smaller in NMOSD patients than in MS patients even after adjusting for age, sex and disease duration (P = 0.002 at T8/T9), whilst there was no difference in cervical CS-SCA between the two diseases. Cervical and thoracic CS-SCA had a negative correlation with EDSS scores in MS patients (P < 0.0001 at C3/C4 and P = 0.0002 at T8/T9) whereas only thoracic CS-SCA correlated with EDSS scores in NMOSD patients (P = 0.0006 at T8/T9). By multiple regression analyses, predictive factors for disability in MS were smaller cervical CS-SCA, progressive course, older age and a higher number of relapses, whilst those in NMOSD were smaller thoracic CS-SCA and older age. CONCLUSIONS: Thoracic CS-SCA is a useful predictive marker for disability in patients with NMOSD whilst cervical CS-SCA is associated with disability in patients with MS.

A Polymorphism rs6726395 in Nrf2 Contributes to the Development of Emphysema-Associated Age in Smokers Without COPD.

INTRODUCTION: Several studies have reported that single nucleotide polymorphisms (SNPs) in the gene encoding NF-E2-related factor 2 (Nrf2) contribute to airflow limitations in smokers without COPD. Although small airway lesions and emphysema contribute cooperatively to airflow limitation, the relationship between Nrf2 SNPs and the development of emphysema in smokers without COPD is not well understood. METHODS: Healthy subjects who underwent an annual health checkup with computed tomography (CT) of the chest at Osaka City University Hospital were prospectively recruited. The percentage of low-attenuation area (%LAA) on chest CT was quantified, and correlations between %LAA, Nrf2 SNP [rs6726395 (G/A)] genotypes, and clinical characteristics were examined. RESULTS: A total of 245 subjects without COPD [non-/light-smoker: 153 (62.4%) and smoker: 92 (37.6%)] were enrolled. The %LAA in the upper lung field was higher than that in the lower lung field (p < 0.001). The %LAA in smokers was significantly higher than that in non-/light-smokers (p = 0.021). The %LAA showed significant but weak correlation with age in all subjects (r = 0.141, p = 0.028). Divided by genotype, the %LAA of the upper lung field was significantly correlated with age in smokers with genotype GG (wild type) (r = 0.333, p = 0.022), but was not significantly correlated with age in smokers with genotype AG/AA. These correlations were not observed in non-/light smokers. CONCLUSION: A polymorphism rs6726395 in Nrf2 can contribute to the development of emphysema-associated aging in smokers. The Nrf2 SNP may be a predictive factor for smoking-induced emphysema, and genotyping of Nrf2 SNP may serve as biomarker for emphysema prevention.

Identification of the Novel Tooth-Specific Transcription Factor AmeloD.

Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs' development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like ( ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell-cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

Oral CD103-CD11b+ classical dendritic cells present sublingual antigen and induce Foxp3+ regulatory T cells in draining lymph nodes.

Sublingual immunotherapy (SLIT) is a safe and efficient treatment for type 1 allergies; however, the underlying immunological mechanisms, particularly the phenotype of oral antigen-presenting cells (APCs) responsible for the induction of regulatory T (Treg) cells, remain unclear. We show here that the sublingual application of ovalbumin (OVA) induced antigen-specific Foxp3+ Treg cells in draining submandibular lymph nodes (ManLNs). Oral APCs were classified into macrophages, classical dendritic cells (cDCs), and Langerhans cells by flow cytometry. A major subset of oral cDCs with the CD103-CD11b+ phenotype showed retinoic acid (RA)-producing activity and converted naive CD4+ T cells to Foxp3+ Treg cells in a transforming growth factor-ß- and RA-dependent manner in vitro. In the ManLNs, migratory CD103-CD11b+ cDCs also showed RA-producing activity. After the sublingual application of fluorescent OVA, fluorescence was detected in oral macrophages in tissues, followed by migratory CD103-CD11b+ cDCs in ManLNs and migratory CD103-CD11b+ cDCs were the main APCs responsible for the induction of sublingual antigen-specific Treg cells. The transfer of OVA-SLIT-induced Treg cells suppressed the OVA-induced hypersensitivity response. These results suggest that oral CD103-CD11b+ cDCs transport sublingual antigens to draining ManLNs and induce antigen-specific Foxp3+ Treg cells, and, thus, provide a rationale for developing cDC-based therapeutic approaches in SLIT.

Immunohistochemical expression of p63, Ki67 and ß-catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder.

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or ß-catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and ß-catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.

Poor glycemic control and decreased renal function are associated with increased intrarenal RAS activity in Type 2 diabetes mellitus.

AIMS: The renin-angiotensin system (RAS) plays an important role in the pathogenesis of diabetic nephropathy. The aim of the present study was to investigate intrarenal RAS activity in patients with type 2 diabetes (T2DM). METHODS: We measured urinary angiotensinogen, a reliable biomarker of intrarenal RAS activity, in 14 controls without T2DM, 25 T2DM patients without nephropathy, 11 chronic kidney disease (CKD) patients without T2DM and 46 CKD patients with T2DM. Associations between urinary angiotensinogen and clinical parameters were examined. RESULTS: Compared with the controls, urinary [angiotensinogen:creatinine] were significantly higher in T2DM patients without nephropathy (4.70 ± 2.22 vs. 8.31 ± 5.27 µg/g, p=0.037). Age, hemoglobin A1c (HbA1c) and fasting plasma glucose correlated significantly and positively with the log{urinary [angiotensinogen:creatinine]} (r=0.632, p=0.007; r=0.405, p=0.027; r=0.583, p=0.003, respectively) in T2DM patients without nephropathy. In contrast, the urinary [angiotensinogen:creatinine] were not significantly different between CKD patients with and without T2DM (22.7 ± 27.8 vs. 33.5 ± 40.8 µg/g, p=0.740); although they were significantly higher when compared with non-CKD patients. In the CKD patients with T2DM systolic blood pressure, serum creatinine, estimated glomerular filtration rate and urinary [albumin:creatinine] correlated significantly with the log{urinary [angiotensinogen:creatinine]} (r=0.412, p=0.004; r=0.308, p=0.037; r=-0.382, p=0.001; r=0.648, p<0.001, p<0.001, respectively). CONCLUSIONS: Our findings indicate that poor glycemic control is significantly associated with intrarenal RAS activity in T2DM patients without nephropathy, and that decreased renal function is significantly associated with intrarenal RAS activity in CKD patients with T2DM.

Significant association of poor glycemic control with increased resistance in efferent arterioles--study of inulin and para-aminohippuric acid clearance in humans.

AIMS: To examine whether glomerular hemodynamic parameters in humans are associated with glycemic control indices, by simultaneously measuring clearance of inulin (Cin) and para-aminohippuric acid (CPHA). METHODS: Thirty-one subjects (age 55.4±14.7 years; 15 men and 16 women; 21 diabetics and 10 non-diabetics) were enrolled. Cin and CPAH were measured simultaneously. Afferent arteriolar resistance (Ra), efferent arteriolar resistance (Re), glomerular hydrostatic pressure (Pglo) and glomerular filtration fraction (FF) were calculated according to Gomez' formula. RESULTS: FF correlated significantly and positively with fasting plasma glucose (FPG), hemoglobin A1c (HbA1c) and glycated albumin (GA) (r=0.396, p=0.0303; r=0.587, p=0.0007; r=0.525, p=0.0070, respectively). Pglo correlated significantly and positively with FPG, HbA1c and GA (r=0.572, p=0.0008; r=0.535, p=0.0019; r=0.540, p=0.0053, respectively). Although there was no significant correlation between Ra and glycemic control indices, Re correlated significantly and positively with HbA1c and GA (r=0.499, p=0.0043; r=0.592, p=0.0018, respectively). FF, Pglo and Re were associated significantly with HbA1c and GA after adjustment for age. CONCLUSIONS: These results demonstrate, in humans, that poor glycemic control is associated with increased Re, but not Ra. It is suggested that increased Re causes increased Pglo, leading to increased FF. Thus, hemodynamic abnormalities with poor glycemic control may be related to glomerular hypertension in humans.

Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.

BACKGROUND AND OBJECTIVE: Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwig's epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS: We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS: Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION: The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.

Divalent cations enhance fluoride binding to Streptococcus mutans and Streptococcus sanguinis cells and subsequently inhibit bacterial acid production.

One preventive effect of topical fluoride application is derived from the fact that fluoride can inhibit bacterial acid production. Furthermore, divalent cations such as Ca(2+) and Mg(2+) increase the binding of fluoride to bacterial cells. These findings suggest that exposure of oral bacteria to fluoride in the presence of divalent cations increases fluoride binding to bacterial cells and subsequently enhances fluoride-induced inhibition of bacterial acid production. This study investigated the effects of fluoride exposure (0-20,000 ppm F) in the presence of Ca(2+) or Mg(2+) prior to glucose challenge on pH fall ability by bacterial sugar fermentation, as well as fluoride binding to bacterial cells by exposure to fluoride, and fluoride release from bacterial cells during bacterial sugar fermentation, using caries-related bacteria, Streptococcus mutans and Streptococcus sanguinis. The pH fall by both streptococci was inhibited by exposure to over 250 ppm F in the presence of Ca(2+) (p < 0.01), whereas in the presence of Mg(2+), the pH fall by S. mutans and S. sanguinis was inhibited after exposure to over 250 and 950 ppm F, respectively (p < 0.05). The amounts of fluoride binding to and released from streptococcal cells increased with the concentration of fluoride the cells were exposed to in the presence of Mg(2+), but were high enough even after 250 ppm F exposure in the presence of Ca(2+). The enhanced inhibition of acid production in the presence of divalent cations is probably due to the improved efficiency of fluoride binding to bacterial cells being improved via these divalent cations.

Glycosphingolipids regulate ameloblastin expression in dental epithelial cells.

Neurotrophin 4 (NT-4) and its receptors regulate the differentiation of ameloblasts in tooth development. Gangliosides, sialic acids that contain glycosphingolipids (GSLs), are involved in a variety of membrane-associated cell physiological functions such as ligand-receptor signal transmission. However, the expression patterns and functions of GSLs during tooth development remain unclear. In this study, we identified strong expressions of GM3 and LacCer in dental epithelium, which give rise to differentiation into enamel-secreting ameloblasts. Exogenous GM3 and LacCer in dental epithelial cells induced the expression of ameloblastin (Ambn), while it was also interesting that GM3 synergistically exerted enhancement of NT-4-mediated Ambn expression. In addition, consistently exogenous GM3 and LacCer in dental epithelial cells induced distinct activation of extracellular signal-regulated kinase 1/2 (ERK1/2), an event upstream of the expression of Ambn. Furthermore, depletion of GSLs from dental epithelial cells by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibited Ambn expression as well as phosphorylation of ERK1/2. In contrast, exogenous addition of GM3 or LacCer rescued the phosphorylation of ERK1/2 repressed by pre-treatment with D-PDMP. Taken together, these results suggest that GM3 and LacCer are essential for NT-4-mediated Ambn expression, and contribute to dental epithelial cell differentiation into ameloblasts.

A phase II study of amrubicin combined with carboplatin for elderly patients with small-cell lung cancer: North Japan Lung Cancer Study Group Trial 0405.

BACKGROUND: Amrubicin, a new anthracycline agent, has shown high activity for small-cell lung cancer (SCLC) in previous studies. However, a combination regimen with amrubicin and platinum has been investigated little. On the basis of previous phase I study, we conducted this study to evaluate the efficacy and the safety of amrubicin and carboplatin for elderly patients with SCLC. METHODS: Chemotherapy-naive elderly patients with SCLC received amrubicin (35 mg/m(2), days 1-3) and carboplatin [area under the curve (AUC) 4.0, day1] every 3 weeks. The primary end point was overall response rate (ORR), and secondary end points were progression-free survival (PFS), overall survival and toxicity profile. RESULTS: From January 2005 to November 2007, 36 patients were enrolled [median age 76 (range 70-83); ECOG performance status of zero and one in 17 and 19 patients, respectively]. One complete response and 31 partial responses were observed (ORR 89%). Median PFS was 5.8 months and median survival time was 18.6 months. Grade 3-4 neutropenia was observed in 97% of the patients and six patients (17%) suffered from grade 3-4 febrile neutropenia. Other toxic effects were moderate and treatment-related death was not observed. CONCLUSIONS: Amrubicin combined with carboplatin is quite effective for SCLC with acceptable toxic effects even for the elderly population. Further evaluation of this regimen is warranted.

Synergistic roles of amelogenin and ameloblastin.

Amelogenin and ameloblastin, the major enamel matrix proteins, are important for enamel mineralization. To identify their synergistic roles in enamel development, we generated Amel X(-/-)/Ambn(-/-) mice. These mice showed additional enamel defects in comparison with Amel X(-/-) or Ambn(-/-) mice. In 7-day-old Amel X(-/-)/Ambn(-/-) mice, not only was the ameloblast layer irregular and detached from the enamel surface, as in Ambn(-/-), but also, the enamel width was significantly reduced in the double-null mice as compared with Amel X(-/-) or Ambn(-/-) mice. Proteomic analysis of the double-null teeth revealed increased levels of RhoGDI (Arhgdia), a Rho-family-specific guanine nucleotide dissociation inhibitor, which is involved in important cellular processes, such as cell attachment. Both Amel X(-/-)/Ambn(-/-) mice and Ambn(-/-) mice displayed positive staining with RhoGDI antibody in the irregularly shaped ameloblasts detached from the matrix. Ameloblastin-regulated expression of RhoGDI suggests that Rho-mediated signaling pathway might play a role in enamel formation.

Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci.

This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of alpha-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of alpha-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding alpha-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of alpha-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of alpha-amylase, the pH fell to 3.9-4.4 and the acid production rate was 0.61-0.92 micromol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, alpha-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of alpha-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. Alpha-amylase inhibitors and xylitol might moderate this activity.

Stenting procedure for sinus stenosis with transverse-sigmoid dural arteriovenous fistulas. A case report.

SUMMARY: We reported the dural AVF case with sinus stenosis, that was entirely treated through the stenting procedure. 61-year-old male had been realizing the attack which causes bilateral visual problem. He would have suffered from the intracranial hypertension caused by dural AVF in the right transverse sinus and left transverse sinus stenosis.We performed TVE and sinus stenting, then used the antiplatelet and the anticoagulant. However, six months later, he suffered from SAH due to recurrence of dural AVF. We performed TVE again, denser packing than usual. Two years later, he have no symptom, angiographically, there was no recurrence of dural AVF and patency of stented sinus. We think denser embolizations should have performed in case of dural AVF with sinus stenting.

Development of a practical immunochromatographic test with recombinant P50 for the diagnosis of Babesia gibsoni infection in dogs.

An immunochromatographic test (ICT), using recombinant truncated P50 (P50t), for the detection of antibodies to Babesia gibsoni was developed and evaluated. Whereas all sera from specific pathogen-free dogs were clearly negative, all sera from dogs experimentally infected with B. gibsoni were clearly positive in the ICT. In addition, the ICT detected no cross-reactivity with sera from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, and B. canis rossi, or with Neospora caninum, and Leishmania infantum. Sequential sera from a dog experimentally infected with B. gibsoni were tested with the ICT; it was shown that the specific antibodies are detectable as early as 6 days post-infection (p.i.) and that strong antibody responses remained until the end of the experiment (144 days p.i.). To evaluate the clinical application of the ICT, a total of 54 serum samples collected from domestic dogs that had been identified as having signs of anaemia at veterinary hospitals in Japan, were tested with the ICT, the previously established enzyme-linked immunosorbent assay (ELISA) and with the indirect fluorescent antibody test (IFAT). Twenty-four of the tested samples (44.4%) were positive in both ICT and ELISA, and (51.8%) in IFAT. The concordance between ELISA and ICT was found to be 100%, and 85.7% with IFAT. Taken together, the results above suggest that the ICT using P50t is rapid, simple, accurate, and suitable for use at clinical sites for the diagnosis of B. gibsoni infection in dogs.