Nuclear receptor 4A1 (NR4A1) upregulated by n-butylidenephthalide via the mitogen-activated protein kinase (MAPK) pathway ameliorates drug-induced gingival enlargement.

Drug-induced gingival enlargement (DIGE) is a side effect of ciclosporin, calcium channel blockers, and phenytoin. DIGE is a serious disease that leads to masticatory and esthetic disorders, severe caries, and periodontitis but currently has no standard treatment. We recently reported that nuclear receptor 4A1 (NR4A1) is a potential therapeutic target for DIGE. This study aimed to evaluate the therapeutic effects of n-butylidenephthalide (BP), which increases the expression of NR4A1, on DIGE. In this study, NR4A1 mRNA expression was analyzed in the patients with periodontal disease (PD) and DIGE. We evaluated the effect of BP on NR4A1 expression in gingival fibroblasts and in a DIGE mouse model. RNA sequencing (RNA-seq) was conducted to identify the mechanisms by which BP increases NR4A1 expression. The results showed that NR4A1 mRNA expression in the patients with DIGE was significantly lower than the patients with PD. BP suppressed the upregulation of COL1A1 expression, which was upregulated by TGF-ß. BP also ameliorated gingival overgrowth in DIGE mice and reduced Col1a1 and Pai1 expression. BP also decreased Il1ß mRNA expression in gingival tissue in DIGE. RNA-seq results showed an increase in the expression of several genes related to mitogen-activated protein kinase including DUSP genes in gingival fibroblasts stimulated by BP. Treatment with ERK and JNK inhibitors suppressed the BP-induced increase in NR4A1 expression. In addition, BP promoted the phosphorylation of ERK in gingival fibroblasts. In conclusion, BP increases NR4A1 expression in gingival fibroblasts through ERK and JNK signaling, demonstrating its potential as a preventive and therapeutic agent against DIGE.

Relationship between CD4+ T-cell counts at baseline and initial periodontal treatment efficacy in patients undergoing treatment for HIV infection: A retrospective observational study.

AIM: To retrospectively investigate the relationship between the CD4+ T-cell counts at baseline and the efficacy of the initial periodontal treatment of patients undergoing treatment for human immunodeficiency virus (HIV) infection using the periodontal inflamed surface area (PISA). MATERIALS AND METHODS: Thirty-three patients with chronic periodontitis who had undergone periodontal examination at baseline and after the initial periodontal treatment were enrolled. PISA was calculated from the periodontal probing depth and bleeding on probing, and the ratio of PISA after treatment to that at baseline (PISA response ratio) was calculated. Groups with a response ratio of <1 and ≥1 were defined as the improvement and the non-improvement groups, respectively. RESULTS: PISA after the initial periodontal treatment significantly decreased compared with that at baseline (p < .05). A weak negative correlation was found between the PISA response ratio and CD4+ T-cell counts at baseline (p < .05). The CD4+ T-cell counts at baseline were significantly higher in the improvement group than in the non-improvement group (p < .05). Multivariate analysis revealed that the CD4+ T-cell counts at baseline was an independent factor that affects the PISA (p < .05). CONCLUSIONS: The higher the CD4+ T-cell counts at baseline in patients undergoing treatment for HIV infection, the more effective the initial periodontal treatment.

The role of nuclear receptor 4A1 (NR4A1) in drug-induced gingival overgrowth.

Drug-induced gingival overgrowth (DIGO) is a side effect of cyclosporine A (CsA), nifedipine (NIF), and phenytoin (PHT). Nuclear receptor 4A1 (NR4A1) plays a role in fibrosis in multiple organs. However, the relationship between NR4A1 and DIGO remains unclear. We herein investigated the involvement of NR4A1 in DIGO. In the DIGO mouse model, CsA inhibited the up-regulation of Nr4a1 expression induced by periodontal disease (PD) in gingival tissue, but not that of Col1a1 and Pai1. We detected gingival overgrowth (GO) in Nr4a1 knock out (KO) mice with PD. A NR4A1 agonist inhibited the development of GO in DIGO model mice. TGF-ß increased Col1a1 and Pai1 expression levels in KO mouse gingival fibroblasts (mGF) than in wild-type mice, while the overexpression of NR4A1 in KO mGF suppressed the levels. NR4A1 expression levels in gingival tissue were significantly lower in DIGO patients than in PD patients. We also investigated the relationship between nuclear factor of activated T cells (NFAT) and NR4A1. NFATc3 siRNA suppressed the TGF-ß-induced up-regulation of NR4A1 mRNA expression in human gingival fibroblasts (hGF). CsA suppressed the TGF-ß-induced translocation of NFATc3 into the nuclei of hGF. Furthermore, NIF and PHT also decreased NR4A1 mRNA expression levels and suppressed the translocation of NFATc3 in hGF. We confirmed that CsA, NIF, and PHT reduced cytosolic calcium levels increased by TGF-ß, while CaCl2 enhanced the TGF-ß-up-regulated NR4A1 expression. We propose that the suppression of the calcium-NFATc3-NR4A1 cascade by these three drugs plays a role in the development of DIGO.

IL-6 Induced by Periodontal Inflammation Causes Neuroinflammation and Disrupts the Blood-Brain Barrier.

Background: Periodontal disease (PD) is a risk factor for systemic diseases, including neurodegenerative diseases. The role of the local and systemic inflammation induced by PD in neuroinflammation currently remains unclear. The present study investigated the involvement of periodontal inflammation in neuroinflammation and blood-brain barrier (BBB) disruption. Methods: To induce PD in mice (c57/BL6), a ligature was placed around the second maxillary molar. Periodontal, systemic, and neuroinflammation were assessed based on the inflammatory cytokine mRNA or protein levels using qPCR and ELISA. The BBB permeability was evaluated by the mRNA levels and protein levels of tight junction-related proteins in the hippocampus using qPCR and immunofluorescence. Dextran tracing in the hippocampus was also conducted to examine the role of periodontal inflammation in BBB disruption. Results: The TNF-α, IL-1ß, and IL-6 levels markedly increased in gingival tissue 1 week after ligation. The IL-6 serum levels were also increased by ligature-induced PD. In the hippocampus, the IL-1ß mRNA expression levels were significantly increased by ligature-induced PD through serum IL-6. The ligature-induced PD decreased the claudin 5 expression levels in the hippocampus, and the neutralization of IL-6 restored its levels. The extravascular 3-kDa dextran levels were increased by ligature-induced PD. Conclusions: These results suggest that the periodontal inflammation-induced expression of IL-6 is related to neuroinflammation and BBB disruption in the hippocampus, ultimately leading to cognitive impairment. Periodontal therapy may protect against neurodegenerative diseases.

Triple A syndrome in Japan.

INTRODUCTION: Triple A syndrome is an autosomal recessive disease, characterized by esophageal achalasia, alacrima, and adrenal insufficiency, as well as involvement of the central, peripheral, and autonomic nervous systems. This disease mimics amyotrophic lateral sclerosis in some patients. The causative gene encodes ALADIN, a nuclear pore complex (NPC) component. Only 5 patients have been reported in Japan. METHODS: We conducted the first nationwide survey of triple A syndrome. Identified mutants were expressed as GFP-fusion proteins in cultured cells. RESULTS: Two new patients were identified, and 1 had a novel mutation (p.Ser182fsX19). All mutant proteins tested were mislocalized from NPC to cytoplasm. CONCLUSIONS: The most consistent neurological manifestation of triple A syndrome in Japanese patients was progressive bulbospinal muscular atrophy with both upper and lower motor neuron involvement, which mimicked motor neuron disease, similar to that seen in patients in Western countries. The identification of the new patients suggests that more cases are undiagnosed in Japan.

Recurrent high-dose intravenous methylprednisolone succinate pulse therapy-induced hepatopathy in a patient with multiple sclerosis.

OBJECTIVE: We describe recurrent and reversible hepatopathy in a girl with multiple sclerosis (MS) after glucocorticoid pulse therapy, to point out the possibility that glucocorticoid may harm the liver. CLINICAL PRESENTATION AND INTERVENTION: An 11-year-old girl with MS, who was treated with high-dose methylprednisolone succinate pulse therapy, developed elevation of liver enzymes. The episodes of hepatopathy occurred 1-5 weeks after the therapy and disappeared within several weeks. The examination for antinuclear antibody and viruses which can cause hepatitis produced negative results. CONCLUSION: The present case emphasizes the possible effects of high-dose glucocorticoids in the induction of liver enzymes and the importance of follow-up liver tests after pulse therapy.

Expression of the IP3R1 promoter-driven nls-lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum.

The cerebellar Purkinje cell monolayer is organized into heterogeneous Purkinje cell compartments that have different molecular compositions. Here we describe a transgenic mouse line, 1NM13, that shows heterogeneous transgene expression in parasagittal Purkinje cell arrays. The transgene consists of a nuclear localization signal (nls) fused to the beta-galactosidase (lacZ) composite gene driven by the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) gene promoter. IP(3)R1-nls-lacZ transgene expression was detected at a single Purkinje cell level over the surface of a whole-mount X-gal-stained cerebellum because of nuclear accumulation of the nls-lacZ activity. Developing cerebella of 1NM13 mice showed stripe-like X-gal staining patterns of parasagittal Purkinje cell subsets. The X-gal stripe pattern was likely determined by an intrinsic property as early as E15 and showed increasing complexity with cerebellar development. The X-gal stripe pattern was reminiscent of, but not identical to, the stripe pattern of zebrin II immunoreactivity. We designated the symmetrical X-gal-positive (transgene-positive, Tg(+)) Purkinje cell stripes about the midline as vermal Tg1(+), Tg2(a, b)(+) and Tg3(a, b)(+) stripes and hemispheric Tg4(a, b)(+), Tg5(a, b)(+), Tg6(a, b, c)(+), and Tg7(a, b)(+) stripes, where a, b, and c indicate substripes. We also assigned three parafloccular substripes Tg8(a, b, c)(+). The boundaries of X-gal stripes at P5 were consistent with raphes in the Purkinje cell layer through which granule cells migrate, suggesting a possible association of the X-gal stripes with raphe formation. Our results indicate that 1NM13 is a good mouse model with a reproducible and clear marker for the compartmentalization of Purkinje cell arrays.

Possible de novo CTG repeat expansion in the DMPK gene of a patient with cardiomyopathy.

CTG triplet repeats of "normal" length in the myotonic dystrophy protein kinase (DMPK) gene have been previously believed to be stable and new pathological expansion was not believed to occur. Here we report possible de novo CTG repeat expansion in the DMPK gene in a patient with cardiomyopathy, who was not diagnosed as having myotonic dystrophy type 1 (DM1) by conventional genetic tests.

Adrenal androgen dehydroepiandrosterone sulfate inhibits vascular remodeling following arterial injury.

Recent epidemiologic studies have suggested that serum dehydroepiandrosterone sulfate (DHEAS) levels have a significant inverse correlation with the incidence of cardiovascular diseases. However, direct evidence for the association with DHEAS and vascular disorders has not yet been explored. DHEAS significantly reduced neointima formation 28 days after surgery without altering other serum metabolite levels in a rabbit carotid balloon injury model. Immunohistochemical analyses revealed the reduction of proliferating cell nuclear antigen (PCNA) index and increase of TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) index, expressing differentiated vascular smooth muscle cell (VSMC) markers in the media 7 days after surgery. In vitro, DHEAS exhibited inhibitory effects on VSMC proliferation and migration activities, inducing G1 cell cycle arrest with upregulation of one of the cyclin dependent kinase (CDK) inhibitors p16(INK4a) and apoptosis with activating peroxisome proliferator-activated receptor (PPAR)-alpha in VSMCs. DHEAS inhibits vascular remodeling reducing neointima formation after vascular injury via its effects on VSMC phenotypic modulation, functions and apoptosis upregulating p16(INK4a)/activating PPARalpha. DHEAS may play a pathophysiological role for vascular remodeling in cardiovascular disease.

[Unexpected treatable dyspnea caused by intratracheal granuloma in an amyotrophic lateral sclerosis patient with mechanical ventilation].

Respiratory insufficiency is a problem that develops in nearly all people diagnosed with amyotrophic lateral sclerosis (ALS). A 46-year-old man with ALS, who had been in a bedridden state with tracheal ventilation support, complained of faintness and dyspnea. The airway pressure of the ventilator had increased, and bleeding from the trachea had occurred several times. A fiberoptic bronchoscopy showed granulation located on the anterior wall of the trachea and severe airway obstruction of the tracheostomy tube. Although a long tracheostomy tube had been intubated for the initial management of the tracheal granulation, a tumor on the posterior tracheal wall had relapsed and occluded the tracheal lumen. A self-expandable metallic airway stent was placed into the tracheal stenosis. After stenting, his symptoms of dyspnea and syncope imploved, and the increased airway pressure of the ventilator was normalized. We speculated that the tracheal granuloma had occurred due to a tracheal mucosal injury related to endotracheal suctioning. We should pay attention to complaints of dyspnea in ALS patients with tracheostomy and make a careful consideration to airway care including suction management.

[A study of bulbar, upper extremity, and lower extremity motor function preservation at the time that respiratory symptoms appear in ALS].

OBJECTIVE: To determine which of the three motor functions (bulbar, upper extremities, lower extremities) was normally preserved at the time that respiratory symptoms occurred in amyotrophic lateral sclerosis (ALS). BACKGROUND: Respiratory failure is the major cause of death in ALS. The course of the disease progression to the onset of subjective respiratory symptom can vary from patient to patient and the diversity of this progression in ALS patients is not well understood. PATIENTS/METHODS: The subjects were 120 Japanese patients with sporadic ALS who were followed-up at our hospital or for whom precise information was available regarding death or the time that the patient required tracheotomy or ventilation assistance. Symptoms started: in the upper limbs in 47 patients; in the lower limbs in 44 patients; in the bulbar region in 22 patients; as the combined type (two regions involved simultaneously on initial presentation) in 6 patients; and with respiratory symptoms in 1 patient. RESULTS: At the time of the appearance of respiratory symptoms, 43 patients (35.8%) had some normally preserved motor function. Significantly fewer patients with progressive bulbar palsy maintained one or more normal motor functions (14%) compared to patients whose disease started in the extremities (43%). Patients were divided into two groups depending on whether they had significant bulbar involvement at the time of the appearance of respiratory symptoms: 88 patients had significant bulbar involvement (B type), and 32 patients did not (R type). Based on the Kaplan-Meier survival curves, no significant difference in the median survival time was noted between the two groups (R type, 29.4 months vs. B type, 32.5 months; p = 0.06). We could not find any difference about clinical characteristics such as gender, onset age, initial site between R and B group. The motor functions that were preserved at the time of respiratory symptom onset included: bulbar function in 32 patients (26.7%); lower extremity function in 12 patients (10%); and upper extremity function in only 3 patients (2.5%). The use of mechanical ventilation in ALS patients with one or more preserved motor functions was significantly higher than in those without any normally preserved motor function (p = 0.03). CONCLUSIONS: From a low rate of upper extremity motor function preservation, respiratory function deterioration appears to be related to upper-limb involvement, likely due to a possible link to diaphragmatic function. Advanced directives should carefully address the use of mechanical ventilation in patients without bulbar symptoms.

Isolated adrenocorticotropic hormone deficiency with transient thyroiditis inducing an adrenal crisis.

OBJECTIVE: It was the aim of this study to describe a patient with isolated adrenocorticotropic hormone deficiency presenting with a variety of involuntary movements who developed an adrenal crisis due to transient thyroiditis. CLINICAL PRESENTATION AND INTERVENTION: A 61-year-old man was hospitalized with a variety of involuntary movements that were suspected manifestations of metabolic encephalopathy. After admission, his general status rapidly deteriorated to a life-threatening condition that included a degree of hyponatremia. The hyponatremia and metabolic encephalopathy provided clues toward a definitive diagnosis. After corticosteroid and sodium supplementation improved the status of the patient, endocrinological examinations revealed that he suffered from isolated adrenocorticotropic hormone deficiency followed by transient thyroiditis that induced an adrenal crisis. CONCLUSION: This case emphasizes the importance of considering hypoadrenalism when encountering hyponatremia or metabolic encephalopathy of unknown etiology.

Biological roles of anti-GM1 antibodies in patients with Guillain-Barré syndrome for nerve growth factor signaling.

To reveal the biological and pathological roles of anti-GM1 antibody in Guillain-Barré syndrome (GBS), we examined its effects on nerve growth factor (NGF) induced TrkA autophosphorylation (NGF-TrkA signaling) in PC12 cells, a sympathetic nerve cell line. The NGF-TrkA signaling is enhanced by exogenous GM1 ganglioside and this phenomenon is regarded as one of the functional aspects of GM1. The IgGs purified from patients' sera inhibited the NGF-TrkA signaling in GM1 pre-incubated PC12 cells. The degrees of inhibition by IgGs from patients paralleled their immunological reactivity to GM1. In addition, the IgGs also inhibited the neurite outgrowth of NGF-treated PC12 cells. Immunoglobulins in the rabbit sera, which were immunized by GM1, also caused a similar suppressive phenomenon. These results suggested that the anti-GM1 antibody could play roles in pathophysiology in anti-GM1 antibody positive GBS through interfering with the neurotrophic action of NGF and GM1 mediated signal modulation including NGF-TrkA signaling. It is suggested that the modulation of GM1 function is one important action of antibodies and could be one of the important mechanisms in GBS.

Physiologically high concentrations of 17beta-estradiol enhance NF-kappaB activity in human T cells.

Estrogen has diverse effects on inflammation and immune responses. That pregnancy is associated with remission of some autoimmune diseases and exacerbation of others suggests that physiological fluctuation in estrogen levels could affect the immune responses in humans. However, the molecular basis for these phenomena is poorly understood. We hypothesized that fluctuations of estrogen levels modulate intracellular signaling for immune responses via estrogen receptors (ERs). In reporter assays, 17beta-estradiol (E2) at a physiologically high concentration increased the activity of NF-kappaB in Jurkat cells stimulated by PMA/ionomycin or TNF-alpha. Overexpression and RNA interference experiments suggested that the effects were mediated through ERbeta. Immunoprecipitation assay showed that both ERalpha and ERbeta are directly associated with NF-kappaB in the cell nucleus. Using chromatin immunoprecipitation assay, we confirmed that ERalpha and ERbeta associated with NF-kappaB and steroid hormone coactivators at the promoter region of NF-kappaB regulated gene. Considering that NF-kappaB regulates the expression of various genes essential for cell growth and death, estrogen could regulate the fate of T cells by affecting the activity of NF-kappaB. To determine whether E2 alters the fate of T cells, we investigated E2 actions on T cell apoptosis, a well-known NF-kappaB-mediated phenomenon. E2 increased apoptosis of Jurkat cells and decreased that of human peripheral blood T cells. Our results indicate that E2 at a physiologically high concentration modulates NF-kappaB signaling in human T cells via ERbeta and affects T cell survival, suggesting that these actions may underlie the gender differences in autoimmune diseases.

Wheelchair economy class syndrome in amyotrophic lateral sclerosis.

A wheelchair-bound 61-year-old diabetic man with amyotrophic lateral sclerosis (ALS) developed sudden respiratory failure. Specific findings for hypoxemia and hypocapnia were incompatible with type II respiratory failure seen in the terminal stages of ALS. 'Economy class syndrome' was diagnosed, with massive thrombosis in the pulmonary arteries and deep vein thrombosis. This case offers a warning for long-term wheelchair users, particularly hypoxemic ALS patients, regarding the risks of treatable pulmonary thromboembolism.

Novel therapeutic strategy for stroke in rats by bone marrow stromal cells and ex vivo HGF gene transfer with HSV-1 vector.

Occlusive cerebrovascular disease leads to brain ischemia that causes neurological deficits. Here we introduce a new strategy combining mesenchymal stromal cells (MSCs) and ex vivo hepatocyte growth factor (HGF) gene transferring with a multimutated herpes simplex virus type-1 vector in a rat transient middle cerebral artery occlusion (MCAO) model. Gene-transferred MSCs were intracerebrally transplanted into the rats' ischemic brains at 2 h (superacute) or 24 h (acute) after MCAO. Behavioral tests showed significant improvement of neurological deficits in the HGF-transferred MSCs (MSC-HGF)-treated group compared with the phosphate-buffered saline (PBS)-treated and MSCs-only-treated group. The significant difference of infarction areas on day 3 was detected only between the MSC-HGF group and the PBS group with the superacute treatment, but was detected among each group on day 14 with both transplantations. After the superacute transplantation, we detected abundant expression of HGF protein in the ischemic brain of the MSC-HGF group compared with others on day 1 after treatment, and it was maintained for at least 2 weeks. Furthermore, we determined that the increased expression of HGF was derived from the transferred HGF gene in gene-modified MSCs. The percentage of apoptosis-positive cells in the ischemic boundary zone (IBZ) was significantly decreased, while that of remaining neurons in the cortex of the IBZ was significantly increased in the MSC-HGF group compared with others. The present study shows that combined therapy is more therapeutically efficient than MSC cell therapy alone, and it may extend the therapeutic time window from superacute to acute phase.

Bone marrow stromal cells that enhanced fibroblast growth factor-2 secretion by herpes simplex virus vector improve neurological outcome after transient focal cerebral ischemia in rats.

BACKGROUND AND PURPOSE: Fibroblast growth factor-2 (FGF-2) administration and bone marrow stromal cell (MSC) transplantation could improve neurological deficits after occlusive cerebrovascular disease. In the present study, we examined the effects of neurological improvement after transient middle cerebral artery occlusion (MCAO) in rats by a novel therapeutic strategy with FGF-2 gene-transferred MSCs by the herpes simplex virus type 1 (HSV-1) vector. METHODS: Adult Wistar rats were anesthetized. Nonmodified MSCs, FGF-2-modified MSCs with HSV-1 1764/-4/pR19/ssIL2-FGF-2, or PBS was administered intracerebrally 24 hours after transient right MCAO. All animals underwent behavioral tests for 21 days, and the infarction volume with 2-3-5-triphenylterazolium was detected 3 days and 14 days after the MCAO. Three days and 7 days after the MCAO, the FGF-2 production in the ipsilateral hemisphere of the MCAO was measured with ELISA. Seven and 14 days after the MCAO, immunohistochemical staining for FGF-2 was applied. RESULTS: The stroke animals receiving FGF-2-modified MSCs demonstrated significant functional recovery compared with the other groups. Fourteen days after the MCAO, there was a significant reduction in infarction volume only in FGF-2-modified MSC-treated group. FGF-2 production in the FGF-2-modified MSC-treated brain was significantly higher compared with the other groups at 3 and 7 days after MCAO. Administrated FGF-2-modified MSCs strongly expressed the FGF-2 protein, which was proven by ELISA. CONCLUSIONS: Our data suggest that the FGF-2 gene-modified MSCs with the HSV-1 vector can contribute to remarkable functional recovery after stroke compared with MSCs transplantation alone.

Postischemic intraventricular administration of FGF-2 expressing adenoviral vectors improves neurologic outcome and reduces infarct volume after transient focal cerebral ischemia in rats.

Fibroblast growth factor (FGF)-2 is a potent neurotrophic and angiogenic peptide. To examine possible protective effects of FGF-2 gene expression against transient focal cerebral ischemia in rats, a replication defective, recombinant adenovirus vector expressing FGF-2, was injected intraventricularly 2 hours after middle cerebral artery occlusion (MCAO). The treatment group showed significant recovery compared with the vehicle-treated groups in terms of serial neurologic severity scores over the 35 days after MCAO. Further, 2,3,5-triphenyltetrazolium chloride staining showed that FGF-2 gene transfer decreased infarct volume by 44% as compared with that in the vehicle-treated groups at 2 days after MCAO. The same tendency of gene transfer effects on infarct volume was confirmed at 35 days after MCAO with hematoxylin/eosin staining. Enzyme-linked immunosorbent assay revealed that FGF-2 concentration was increased significantly at 2 days after MCAO, not only in cerebrospinal fluid but also in cerebral substance in the lesioned and treated animals. These results suggested that FGF-2 gene transfer using these adenoviral vectors might be a useful modality for the treatment of occlusive cerebrovascular disease even after the onset of stroke.