Prevalence, Clinical Profiles, and Prognosis of Stiff-Person Syndrome in a Japanese Nationwide Survey.

BACKGROUND AND OBJECTIVES: To elucidate current epidemiologic, clinical, and immunologic profiles and treatments of stiff-person syndrome (SPS) in Japan. METHODS: A nationwide mail survey was conducted using an established method. Data processing sheets were sent to randomly selected departments of internal medicine, neurology, pediatrics, psychiatry, and neurosurgery in hospitals and clinics throughout Japan to identify patients with SPS who were seen between January 2015 and December 2017. RESULTS: Thirty cases were identified as glutamic acid decarboxylase 65 (GAD65)-positive SPS cases on the basis of detailed clinical data of 55 cases. Four patients had α1 subunit of glycine receptor (GlyR) antibodies, and 1 patient had both GAD65 and GlyR antibodies. The total estimated number of patients with GAD65-positive SPS was 140, and the estimated prevalence was 0.11 per 100,000 population. The median age at onset was 51 years (range, 26-83 years), and 23 (76%) were female. Of these, 70% had classic SPS, and 30% had stiff-limb syndrome. The median time from symptom onset to diagnosis was significantly longer in the high-titer GAD65 antibody group than in the low-titer group (13 months vs 2.5 months, p = 0.01). The median modified Rankin Scale (mRS) at baseline was 4, and the median mRS at the last follow-up was 2. Among the 29 GAD65-positive patients with ≥1 year follow-up, 7 received only symptomatic treatment, 9 underwent immunotherapy without long-term immunotherapy, and 13 received long-term immunotherapy such as oral prednisolone. The coexistence of type 1 diabetes mellitus and the lack of long-term immunotherapy were independent risk factors for poor outcome (mRS ≥3) in the GAD65-positive patients (odds ratio, 15.0; 95% CI 2.6-131.6; p = 0.001; odds ratio, 19.8; 95% CI 3.2-191.5; p = 0.001, respectively). DISCUSSION: This study provides the current epidemiologic and clinical status of SPS in Japan. The symptom onset to the diagnosis of SPS was longer in patients with high-titer GAD65 antibodies than in those with low-titer GAD65 antibodies. The outcome of patients with SPS was generally favorable, but more aggressive immunotherapies are necessary for GAD65-positive patients with SPS.

[Stiff-Person Syndrome].

Stiff-person syndrome (SPS) is a rare autoimmune neurological disorder characterized by progressive axial muscle stiffness, central nervous system hyper-excitability, and painful stimulus-sensitive muscle spasms. SPS is classified into classic SPS and SPS variants, including stiff-limb syndrome (SLS) and progressive encephalomyelitis with rigidity and myoclonus (PERM), based on clinical presentation. SPS responds to immunotherapy, and several autoantigens have been identified. Most patients with SPS have high-titers of antibodies against glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of γ-aminobutyric acid (GABA), and up to 15% of the patients have antibodies against the glycine receptor α-subunit.

Clinical Activity and Exploratory Resistance Mechanism of Milademetan, an MDM2 Inhibitor, in Intimal Sarcoma with MDM2 Amplification: An Open-Label Phase Ib/II Study.

Intimal sarcoma is an extremely rare, life-threatening malignant neoplasm. Murine double minute 2 (MDM2) amplification is observed in >70% of intimal sarcomas. Milademetan, an MDM2 inhibitor, may provide clinical benefit in this patient population. We conducted a phase Ib/II study in patients with MDM2-amplified, wild-type TP53 intimal sarcoma as a substudy of a large nationwide registry for rare cancers in Japan. Milademetan (260 mg) was administered orally once daily for 3 days every 14 days, twice in a 28-day cycle. Of 11 patients enrolled, 10 were included in the efficacy analysis. Two patients (20%) showed durable responses for >15 months. Antitumor activity correlated with TWIST1 amplification (P = 0.028) and negatively with CDKN2A loss (P = 0.071). Acquired TP53 mutations were detected in sequential liquid biopsies as a novel exploratory resistance mechanism to milademetan. These results suggest that milademetan could be a potential therapeutic strategy for intimal sarcoma. SIGNIFICANCE: Strategies to optimize outcomes could include the use of new biomarkers (TWIST1 amplification and CDKN2A loss) to select patients with MDM2-amplified intimal sarcoma who might benefit from milademetan and combination with other targeted treatments. Sequential liquid biopsy of TP53 can be used to evaluate disease status during treatment with milademetan. See related commentary by Italiano, p. 1765. This article is highlighted in the In This Issue feature, p. 1749.

Pulmonary cancers across different histotypes share hybrid tuft cell/ionocyte-like molecular features and potentially druggable vulnerabilities.

Tuft cells are chemosensory epithelial cells in the respiratory tract and several other organs. Recent studies revealed tuft cell-like gene expression signatures in some pulmonary adenocarcinomas, squamous cell carcinomas (SQCC), small cell carcinomas (SCLC), and large cell neuroendocrine carcinomas (LCNEC). Identification of their similarities could inform shared druggable vulnerabilities. Clinicopathological features of tuft cell-like (tcl) subsets in various lung cancer histotypes were studied in two independent tumor cohorts using immunohistochemistry (n = 674 and 70). Findings were confirmed, and additional characteristics were explored using public datasets (RNA seq and immunohistochemical data) (n = 555). Drug susceptibilities of tuft cell-like SCLC cell lines were also investigated. By immunohistochemistry, 10-20% of SCLC and LCNEC, and approximately 2% of SQCC expressed POU2F3, the master regulator of tuft cells. These tuft cell-like tumors exhibited "lineage ambiguity" as they co-expressed NCAM1, a marker for neuroendocrine differentiation, and KRT5, a marker for squamous differentiation. In addition, tuft cell-like tumors co-expressed BCL2 and KIT, and tuft cell-like SCLC and LCNEC, but not SQCC, also highly expressed MYC. Data from public datasets confirmed these features and revealed that tuft cell-like SCLC and LCNEC co-clustered on hierarchical clustering. Furthermore, only tuft cell-like subsets among pulmonary cancers significantly expressed FOXI1, the master regulator of ionocytes, suggesting their bidirectional but immature differentiation status. Clinically, tuft cell-like SCLC and LCNEC had a similar prognosis. Experimentally, tuft cell-like SCLC cell lines were susceptible to PARP and BCL2 co-inhibition, indicating synergistic effects. Taken together, pulmonary tuft cell-like cancers maintain histotype-related clinicopathologic characteristics despite overlapping unique molecular features. From a therapeutic perspective, identification of tuft cell-like LCNECs might be crucial given their close kinship with tuft cell-like SCLC.

Application of ultrasound in a case of eosinophilic fasciitis mimicking stiff-person syndrome.

Eosinophilic fasciitis (EF) is a rare disorder characterized by muscle stiffness mimicking other neuromuscular diseases. The diagnosis of EF is made on the basis of typical skin lesions. We report a case of a 36-year-old male patient with suspected stiff-person syndrome (SPS), who presented with progressive limb muscle stiffness and limited mobility of both wrists without obvious skin changes. Ultrasound revealed fascial thickening of bilateral upper and lower limb muscles and enlargement of hypoechoic tissues around the flexor digitorum tendons of the wrist. Skin and fascia biopsy confirmed the diagnosis of EF. Prednisolone therapy resulted in the improvement of muscle stiffness and tightness. Our findings suggest the need to consider connective tissue diseases such as EF in a patient with atypical features of SPS. Ultrasound is helpful for visualizing the causes of muscle stiffness and joint contractures in EF patients.

Multicenter phase II trial of trastuzumab deruxtecan for HER2-positive unresectable or recurrent biliary tract cancer: HERB trial.

Trastuzumab deruxtecan (DS-8201) is an antibody-drug conjugate composed of a humanized monoclonal anti-HER2 antibody, a cleavable tetrapeptide-based linker and a potent topoisomerase I inhibitor. The drug's efficacy has been proven in HER2-positive breast and gastric cancers. The rate of HER2 positivity in biliary tract cancer (BTC) has been reported to be 5-20%, and case reports and clinical trials have suggested that HER2 inhibitors might be active in HER2-positive BTC. Here we describe the rationale and design of the phase II HERB trial that will evaluate the efficacy and safety of trastuzumab deruxtecan in patients with HER2-expressing unresectable or recurrent BTC. The primary end point will be the centrally assessed objective response rate in HER2-positive patients.

Trastuzumab deruxtecan (DS-8201) is a new drug against HER2, a receptor on cell membranes that has sensitivity to targeted inhibitors. The drug's efficacy has been proven in HER2-positive breast and gastric cancers. Some studies have suggested that HER2 inhibitors might be active in HER2-positive biliary tract cancers. This article describes the design of a new clinical trial. The HERB trial is designed to evaluate the efficacy and safety of trastuzumab deruxtecan in patients with HER2-expressing biliary tract cancers. Clinical trial registration: JMA-IIA00423.

Complex hereditary peripheral neuropathies caused by novel variants in mitochondrial-related nuclear genes.

Mitochondrial disorders are a group of clinically and genetically heterogeneous multisystem disorders and peripheral neuropathy is frequently described in the context of mutations in mitochondrial-related nuclear genes. This study aimed to identify the causative mutations in mitochondrial-related nuclear genes in suspected hereditary peripheral neuropathy patients. We enrolled a large Japanese cohort of clinically suspected hereditary peripheral neuropathy patients who were mutation negative in the prescreening of the known Charcot-Marie-Tooth disease-causing genes. We performed whole-exome sequencing on 247 patients with autosomal recessive or sporadic inheritance for further analysis of 167 mitochondrial-related nuclear genes. We detected novel bi-allelic likely pathogenic/pathogenic variants in four patients, from four mitochondrial-related nuclear genes: pyruvate dehydrogenase beta-polypeptide (PDHB), mitochondrial poly(A) polymerase (MTPAP), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, beta subunit (HADHB), and succinate-CoA ligase ADP-forming beta subunit (SUCLA2). All these patients showed sensory and motor axonal polyneuropathy, combined with central nervous system or multisystem involvements. The pathological analysis of skeletal muscles revealed mild neurogenic changes without significant mitochondrial abnormalities. Targeted screening of mitochondria-related nuclear genes should be considered for patients with complex hereditary axonal polyneuropathy, accompanied by central nervous system dysfunctions, or with unexplainable multisystem disorders.

Intrathymic Plasmablasts Are Affected in Patients With Myasthenia Gravis With Active Disease.

BACKGROUND AND OBJECTIVES: To investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact. METHODS: Thymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19+B220high thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated. RESULTS: The frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19+B220high thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy. DISCUSSION: Our findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG.

[Stiff-Person Syndrome].

Stiff-person syndrome (SPS) is a neurological disorder characterized by fluctuating muscle rigidity and painful spasms that occur spontaneously or are triggered by diverse stimuli. Partial or segmental forms of the disorder, such as stiff-limb syndrome (SLS) and the more severe disease called progressive encephalomyelitis with rigidity and myoclonus (PERM), are usually considered within the spectrum of SPS. SPS responds to immunotherapies, and several autoantigens have been identified. Most patients with SPS have high titers of antibodies against glutamic acid decarboxylase (GAD), the enzyme that limits the rate of the synthesis of γ-aminobutyric acid (GABA), and up to 15% have antibodies against the glycine receptor α-subunit.

Micro-CT assessment of the effect of silver diammine fluoride on inhibition of root dentin demineralization.

This study evaluated the ability of different types of silver diammine fluoride (SDF) to inhibit dentin demineralization using micro-focused X-ray computed-tomography (µCT). Dentin specimens were divided into five groups (n=10); no-treatment (control), 3.8% SDF (RC), 38% SDF, 38% SDF with potassium iodide (SDF/KI), and potassium fluoride (KF). The treated-dentin surfaces were subjected to demineralization for 7-days and assessed using µCT to determine mineral loss (ML) values. Specimens were also analyzed with scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The ML values of the SDF and KF groups were significantly lower than those of the RC and SDF/KI groups. EDS detected fluoride ions in the SDF and KF groups but not in the RC and SDF/KI groups. It was concluded that 38% SDF demonstrated a high ability to inhibit dentin demineralization while additional application of KI may diminish the inhibitory effect of SDF. The amount of dentin demineralization with SDF treatments was material dependent.

Effects of silver diamine fluoride preparations on biofilm formation of Streptococcus mutans.

Effects of silver diamine fluoride preparations (SDFs) on cariogenic biofilm formation on root dentin (RD) were investigated. Streptococcus mutans (S. mutans) biofilms were formed on bovine RD blocks coated with one of three the SDFs (38%-SDF, 3.8%-SDF and 35%-SDF+potassium-iodide; SDF+KI) and a non-coated Control which were quantified (spectrometric-measurement) and thickness measured (optical coherence tomography) after 20 h. Bacterial viability test (BacLight) and biofilm-morphometry (SEM) of 2 h biofilms were also performed. The amounts of biofilms (bacteria and water insoluble glucan) and the thickness of biofilm were minimum on 38%-SDF specimen; 3.8%-SDF and SDF+KI had significantly more than that, but had significantly less than Control (p<0.05). Most S. mutans cells found dead and morphology damaged by 38%-SDF. Some dead bacteria and remarkably damaged biofilms were observed in case of 3.8%-SDF and SDF+KI. Inhibition potential of 3.8%-SDF and SDF+KI on S. mutans biofilm formation is almost similar, although not equivalent to 38%-SDF.

Effect of antioxidant supplementation on skeletal muscle and metabolic profile in aging mice.

Aging induces drastic changes in muscle mass and function (sarcopenia); however, the detailed mechanisms underlying sarcopenia remain poorly understood. Recent studies suggested that age-related increases in oxidative stress induce muscle atrophy. In this study, we investigated the effect of 6-month supplementation of antioxidants, specifically piceatannol (PIC) and enzymatically modified isoquercitrin (EMIQ), on age-related physiological changes, including skeletal muscle weight and quality, in 25-month-old (OLD) mice, compared to in 4-month-old (young, YNG) C57BL/6J mice. Muscle weight corrected by body weight significantly declined in OLD mice, compared to in YNG mice. The control OLD mice also showed changes in the expression of genes related to muscle fiber type, reduced locomotor activity, and increased oxidative stress markers in blood. Consistent with the muscle weight and quality changes, whole-body fat oxidation during sedentary conditions and exercise periods in control OLD mice was significantly lower than that in YNG mice. Interestingly, compared to the control OLD mice, the PIC- or EMIQ-fed OLD mice showed higher fat oxidation. Furthermore, EMIQ, but not PIC, increased locomotor activity, the expression of genes encoding antioxidant enzymes, and suppressed the carbonylated protein in the skeletal muscle of OLD mice. These results suggested that chronic antioxidant intake could alleviate aging-related muscle function changes.

Prediction of improvement after extended thymectomy in non-thymomatous myasthenia gravis patients.

BACKGROUND: It is popularly believed that myasthenia gravis (MG) patients show acetylcholine receptor antibody (AChRAb) production associated with the thymus (germinal centers, approximately 80%). It has been suggested that thymectomy can remove the area of autoantibody production. This study aimed to determine whether the solid volume of the thymus calculated using three-dimensional (3D) imaging could be used to predict the efficacy of thymectomy. Additionally, the study assessed the relationships of the solid volume with germinal centers, change in the serum AChRAb level, postoperative MG improvement, and prednisolone (PSL) dose reduction extent. METHODS: This retrospective study included 12 consecutive non-thymomatous MG patients (9 female and 3 male patients), who underwent extended thymectomy at our institution over the last 10 years. The mean patient age was 43.3 ± 14.2 years (range, 12-59 years). The study assessed the number of germinal centers per unit area, change in the serum AChRAb level, postoperative MG improvement, PSL dose reduction extent, and solid volume of the thymus. RESULTS: The number of germinal centers per unit area was significantly correlated with the solid volume of the thymus. The PSL dose reduction extent tended to be correlated with the solid volume. CONCLUSIONS: Our findings suggest that the solid volume of the thymus can possibly predict steroid dose reduction. Additionally, the solid volume of the thymus in 3D images is the most important indicator for predicting the efficacy of extended thymectomy.

Effect of two bleaching regimens on enamel bonding performance.

This study evaluated the post-bleaching bonding performance of a one-step and two-step self-etch adhesive (SEA) to bleached enamel using in-office or at-home bleaching. Bovine enamel surfaces were treated with an in-office or at-home bleaching agent and divided into four post-bleach time periods: immediate, one day (1d), one week (1w) and one month (1m) before application of SEA systems. Enamel was bonded using the following procedures: Group 1: one-step SEA application (BeautiBond Multi, BBM), Group 2: two-step SEA application (FL-Bond II, FLBII). Microshear bond strengths (µSBS) were measured 24-h after bonding for each of the post-bleaching time periods. Acid-base resistant zones (ABRZs) were observed under SEM. Data were analyzed using three-way ANOVA. Bleaching caused a significant decrease of the immediate µSBSs for both adhesives (p≤0.005) then increased. Thick ABRZs were observed in BBM and FLBII after bleaching. Enamel µSBSs were adversely affected by bleaching. Thick ABRZ was created with bleaching.

Effect of silver diammine fluoride application on dentin bonding performance.

The purpose of this study was to evaluate the effects of two different concentrations of silver diammine fluoride (SDF) on dentin bonding performance. Human mid-coronal dentin was treated with either distilled water (control), 3.8%SDF or 38%SDF for 3 min. A two-step self-etch adhesive and resin composite were applied according to manufacturer's instructions. After thermocycling (TC) at 0, 5,000 and 10,000 cycles, microtensile bond strength (µTBS) testing and morphological assessment of resin-dentin bonding interface were performed. At 0 TC, µTBSs of 3.8% and 38%SDF were significantly reduced (p<0.05). At 5,000 and 10,000 TC, µTBSs of 3.8%SDF were comparable to those of the control group, whereas the µTBSs of 38%SDF were significantly lower (p<0.05). Acid-base resistance zone formation was observed in all groups, however, slope-shaped formation was identified only in the SDF groups. The µTBSs and interfacial morphology were influenced by concentration of SDF and also TC.

Morphological and elemental analysis of silver penetration into sound/demineralized dentin after SDF application.

OBJECTIVE: The aim of this study was to evaluate the penetration depth of silver into sound and demineralized dentin after application of silver diamine fluoride (SDF). METHODS: Two hundred and eighty-eight dentin specimens were used. The specimens were divided into 3 groups: (1) sound dentin (control), (2) 30min EDTA-treated dentin; and (3) 13h EDTA-treated dentin. SDF was applied to all specimens. Each group was divided into 3 subgroups according to storage time into: 24h, 2 weeks and 1-year storage time. Each subgroup was further divided into four subgroups (n=8) according to different examinations as optical microscope (OM) observation, scanning electron microscopic (SEM) observation, elemental analysis with energy dispersive spectroscopy (EDS) and Micro-PIXE test. RESULTS: The OM showed discoloration in the superficial layer after 24h and keep extending deeper after 2 weeks and 1-year. SEM showed silver crystals within dentinal tubules after 2 weeks and 1-year. EDS analysis can detect silver penetration only in the 1-year group reaching around 1200µm inside dentin. Micro-PIXE test detected silver at all time intervals, confirming the EDS depth results. SIGNIFICANCE: It can be concluded that silver ions can completely infiltrate the demineralized dentin lesion with further penetration into the underlying mineralized dentin.

The updated retrospective questionnaire study of sporadic inclusion body myositis in Japan.

BACKGROUND: Sporadic inclusion body myositis (sIBM) is the most prevalent muscle disease in elderly people, affecting the daily activities. sIBM is progressive with unknown cause and without effective treatment. In 2015, sIBM was classified as an intractable disease by the Japanese government, and the treatment cost was partly covered by the government. This study aimed to examine the changes in the number of patients with sIBM over the last 10 years and to elucidate the cross-sectional profile of Japanese patients with sIBM. METHODS: The number of sIBM patients was estimated through a reply-paid postcard questionnaire for attending physicians. Only patients diagnosed as "definite" or "probable" sIBM by clinical and biopsy sIBM criteria were included in this study (Lancet Neurol 6:620-631, 2007, Neuromuscul Disord 23:1044-1055, 2013). Additionally, a registered self-administered questionnaire was also sent to 106 patients who agreed to reply via their attending physician, between November 2016 and March 2017. RESULTS: The number of patients diagnosed with sIBM for each 5-year period was 286 and 384 in 2011 and 2016, respectively. Inability to stand-up, cane-dependent gait, inability to open a plastic bottle, choking on food ingestion, and being wheelchair-bound should be included as sIBM milestones. Eight patients were positive for anti-hepatitis C virus antibody; three of them were administered interferon before sIBM onset. Steroids were administered to 33 patients (31.1%) and intravenous immunoglobulin to 46 patients (43.4%). From 2016 to 2017, total of 70 patients applied for the designated incurable disease medical expenses subsidy program. Although the treatment cost was partly covered by the government, many patients expressed psychological/mental and financial anxieties. CONCLUSIONS: We determined the cross-sectional profile of Japanese patients with sIBM. Continuous support and prospective surveys are warranted.

Effect of Office Bleaching on Enamel Bonding Performance.

PURPOSE: To evaluate the effect of in-office bleaching on enamel microshear bond strength (µSBS) of two self-etch adhesives (SEA). MATERIALS AND METHODS: For the bleaching group, bovine enamel surfaces were treated with a bleaching agent and divided into two main groups: one in which adhesive was applied immediately after bleaching and another in which adhesive was applied after one week. Enamel was bonded using the following procedures: group 1: application of a two-step SEA (Clearfil SE Bond 2, SE2, Kuraray Noritake); group 2: etching with 40% phosphoric acid (PA) and application of SE2;.group 3: application of a one-step SEA (G-Premio Bond, GP, GC); group 4: etching with PA and application of GP. Resin composite was placed and µSBS was measured at 24 h (immediately) and 1 week after bleaching. Acid-base resistant zones (ABRZ) were observed under SEM. Three-way ANOVA was used for data analysis. RESULTS: Bleaching significantly decreased the µSBSs even after PA etching with both SE2 and GP (p ≤ 0.005). However, there were no significant differences in µSBS between groups with and without PA etching, except for SE2 without bleaching, in which the µSBS significantly increased with PA etching (p < 0.005). Thick ABRZs were found in SE2 and GP after bleaching, which exhibited an irregular wave-like shape for PA-treated specimens. CONCLUSION: The enamel µSBSs were adversely affected by in-office bleaching. Thick ABRZs were created with bleaching and PA treatment.

Effect of fluoride mouthrinse and fluoride concentration on bonding of a one-step self-etch adhesive to bovine root dentin.

This study investigated the effect of fluoride mouthrinses on bonding of a one-step self-etch adhesive to bovine root dentin. Application of a NaF solution (0, 450, 900, or 9,000 ppm) to bovine root dentin surfaces was performed for 30 s (immediate) or before bonding of an all-in-one adhesive. Microtensile bond strength (µTBS) testing and scanning electron microscopic (SEM) observation of the acid-base resistance zone (ABRZ) were performed. µTBS values and calculated ABRZ areas were analyzed by using two-way ANOVA and the t-test with Bonferroni correction. The significance level was set at P = 0.05 (n = 30). Fracture mode was analyzed with the Mann-Whitney U test with Bonferroni correction. The significance level set at P = 0.00178 (n = 30). µTBS did not significantly differ between the control, 450 ppm F, and 900 ppm F specimens (P > 0.05) but was significantly lower in 9,000 ppm F specimens (P < 0.05). SEM showed a significant increase in acid resistance at the adhesive-dentin interface, including the ABRZ, after fluoride application.