A Novel Method to Differentiate Tonsil-Derived Mesenchymal Stem Cells In Vitro into Estrogen-Secreting Cells

BACKGROUND@#The advantages of tonsil-derived mesenchymal stem cells (TMSCs) over other mesenchymal stem cells (MSCs) include higher proliferation rates, various differentiation potentials, efficient immune-modulating capacity, and ease of obtainment. Specifically, TMSCs have been shown to differentiate into the endodermal lineage. Estrogen deficiency is a major cause of postmenopausal osteoporosis and is associated with higher incidences of ischemic heart disease and cerebrovascular attacks during the postmenopausal period. Therefore, stem cell-derived, estrogen-secreting cells might be used for estrogen deficiency. @*METHODS@#Here, we developed a novel method that utilizes retinoic acid, insulin-like growth factor-1, basic fibroblast growth factor, and dexamethasone to evaluate the differentiating potential of TMSCs into estrogen-secreting cells. The efficacy of the novel differentiating method for generation of estrogen-secreting cells was also evaluated with bone marrow- and adipose tissue-derived MSCs. @*RESULTS@#Incubating TMSCs in differentiating media induced the gene expression of cytochrome P450 19A1 (CYP19A1), which plays a key role in estrogen biosynthesis, and increased 17b-estradiol secretion upon testosterone addition. Furthermore, CYP11A1, CYP17A1, and 3b-hydroxysteroid dehydrogenase type-1 gene expression levels were significantly increased in TMSCs. In bone marrow-derived and adipose tissue-derived MSCs, this differentiation method also induced the gene expression of CYP19A1, but not CYP17A1, suggesting TMSCs are a superior source for estrogen secretion. @*CONCLUSION@#These results imply that TMSCs can differentiate into functional estrogen-secreting cells, thus providing a novel, alternative cell therapy for estrogen deficiency.

A Novel Method to Differentiate Tonsil-Derived Mesenchymal Stem Cells In Vitro into Estrogen-Secreting Cells

BACKGROUND@#The advantages of tonsil-derived mesenchymal stem cells (TMSCs) over other mesenchymal stem cells (MSCs) include higher proliferation rates, various differentiation potentials, efficient immune-modulating capacity, and ease of obtainment. Specifically, TMSCs have been shown to differentiate into the endodermal lineage. Estrogen deficiency is a major cause of postmenopausal osteoporosis and is associated with higher incidences of ischemic heart disease and cerebrovascular attacks during the postmenopausal period. Therefore, stem cell-derived, estrogen-secreting cells might be used for estrogen deficiency. @*METHODS@#Here, we developed a novel method that utilizes retinoic acid, insulin-like growth factor-1, basic fibroblast growth factor, and dexamethasone to evaluate the differentiating potential of TMSCs into estrogen-secreting cells. The efficacy of the novel differentiating method for generation of estrogen-secreting cells was also evaluated with bone marrow- and adipose tissue-derived MSCs. @*RESULTS@#Incubating TMSCs in differentiating media induced the gene expression of cytochrome P450 19A1 (CYP19A1), which plays a key role in estrogen biosynthesis, and increased 17b-estradiol secretion upon testosterone addition. Furthermore, CYP11A1, CYP17A1, and 3b-hydroxysteroid dehydrogenase type-1 gene expression levels were significantly increased in TMSCs. In bone marrow-derived and adipose tissue-derived MSCs, this differentiation method also induced the gene expression of CYP19A1, but not CYP17A1, suggesting TMSCs are a superior source for estrogen secretion. @*CONCLUSION@#These results imply that TMSCs can differentiate into functional estrogen-secreting cells, thus providing a novel, alternative cell therapy for estrogen deficiency.

Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis

Background@#Fabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear. @*Methods@#Renal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys. @*Results@#Compared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice. @*Conclusion@#These findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis

Background@#Fabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear. @*Methods@#Renal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys. @*Results@#Compared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice. @*Conclusion@#These findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Cigarette Smoking Preferentially Affects Intracranial Vessels in Young Males: A Propensity-Score Matching Analysis / 신경중재치료의학

PURPOSE: Cigarette smoking (CS) is one of the major risk factors of cerebral atherosclerotic disease, however, its level of contribution to extracranial and intracranial atherosclerotic stenosis (ECAS and ICAS) was not fully revealed yet. The purpose of our study was to assess the association of CS to cerebral atherosclerosis along with other risk factors. MATERIALS AND METHODS: All consecutive patients who were angiographically confirmed with severe symptomatic cerebral atherosclerotic disease between January 2002 and December 2012 were included in this study. Multivariate logistic regression analyses were performed to identify risk factors for ECAS and ICAS. Thereafter, CS group were compared to non-CS group in the entire study population and in a propensity-score matched population with two different age-subgroups. RESULTS: Of 1709 enrolled patients, 794 (46.5%) had extracranial (EC) lesions and the other 915 (53.5%) had intracranial (IC) lesions. CS group had more EC lesions (55.8% vs. 35.3%, P<0.001) whereas young age group (<50 years) had more IC lesion (84.5% vs. 47.6%, P<0.001). In multivariate analysis, seven variables including CS, male, old age, coronary heart disease, higher erythrocyte sedimentation rate, multiple lesions, and anterior lesion were independently associated with ECAS. In the propensity-score matched CS group had significant more EC lesion compared to non-CS group (65.7% vs. 47.9%) only in the old age subgroup. CONCLUSION: In contrast to a significant association between CS and severe symptomatic ECAS shown in old population, young patients did not show this association and showed relatively higher preference of ICAS.

Development of cell models for high-throughput screening system of Charcot-Marie-Tooth disease type 1

PURPOSE: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. MATERIALS AND METHODS: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. RESULTS: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. CONCLUSION: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

Dynamic Transcriptional Events in Distal Sural Nerve Revealed by Transcriptome Analysis

Compared with biochemical information available about the diseases in the central nervous system, that for peripheral neuropathy is quite limited primarily due to the difficulties in obtaining samples. Characterization of the core pathology is a prerequisite to the development of personalized medicine for genetically heterogeneous diseases, such as hereditary motor and sensory neuropathy (HMSN). Here, we first documented the transcriptome profile of distal sural nerve obtained from HMSN patients. RNA-seq analysis revealed that over 12,000 genes are expressed in distal sural nerve. Among them 4,000 transcripts are novel and 10 fusion genes per sample were observed. Comparing dataset from whole exome sequencing revealed that over 1,500 transcriptional base modifications occur during transcription. These data implicate that dynamic alterations are generated when genetic information are transitioned in distal sural nerve. Although, we could not find significant alterations associated with HMSN, these data might provide crucial information about the pathophysiology of HMSN. Therefore, next step in the development of therapeutic strategy for HMSN might be unveiling biochemical and biophysical abnormalities derived from those potent variation.

Genome-wide scan of granular corneal dystrophy, type II: confirmation of chromosome 5q31 and identification of new co-segregated loci on chromosome 3q26.3

Granular corneal dystrophy, type II (CGD2; Avellino corneal dystrophy) is the most common corneal dystrophy among Koreans, but its pathophysiology is still poorly understood. Many reports showed that even though the causative mutation is the same TGFBI R124H mutation, there are severe and mild phenotypes of the corneal dystrophy. We also observed the phenotype differences in our samples. For this reason, we focused our effort on the identification of unknown genetic factor related to phenotype variation. A total 551 individuals from 59 families were genotyped with SNP chip and used in genome-wide linkage analysis. From single-point linkage analyses, we confirmed the known 5q31 region for TGFBI gene, and selected novel nine candidate loci for CGD2. In simulation analysis, the only 3q26.3 region including neuroligin 1 gene (NLGN1) was supported by empirical statistic significance. To investigate the effect of genetic heterogeneity in linkage analysis, we classified CGD2 families into two subgroups. Although we could not find a significant evidence for correlation between the 3q26.3 region and CGD2 phenotypes, this first genome-wide analysis with CGD2 families in Korea has a very important value for offering insights in genetics of CGD2. In addition, the co-segregating loci with CGD2 including 3q26.3 would be a good target for further study to understand the pathophysiology of CGD2.

Tissue-specific activation of mitogen-activated protein kinases for expression of transthyretin by phenylalanine and its metabolite, phenylpyruvic acid

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.

Comparision in the yield of fetal nucleated red blood cell between the first-and second-trimester using double density gradient centrifugation / 대한산부인과학회지

OBJECTIVE: The aim of our study was to make a practical comparative evaluation of the first and second trimesters in order to determine the period during which a higher yield of fetal nucleated red blood cells (FNRBCs) can be obtained. METHODS: NRBCs were isolated from maternal blood during the first and second trimesters of pregnancy using double Percoll gradients with different osmolarities. Magnetic activated cell sorting was performed with Kleihauer-Betke stain. We isolated fetal NRBCs from 10 mL of samples of maternal blood and determined fetal sex and fetal aneuploidy by fluorescence in situ hybridization (FISH). RESULTS: The average number of NRBCs was 9.85 in samples obtained during the first trimester and 14.88 in samples obtained during the second trimester (P=0.07). The average number of NRBCs with Y chromosome signals was 5.73 in the first trimester and 8.22 in second trimester (P=0.56). However, the percentage of NRBCs with Y chromosome signals in the first trimester (70.6%) was significantly higher than in the second trimester (59.8%) (P=0.049). We diagnosed the blood samples from 7 pregnant women having fetal aneuploidy using this method and the number of NRBCs was 18.4. CONCLUSION: The method using Percoll osmolarity and a double density gradient system may be a very useful method for separation of NRBCs in the first trimester of pregnancy and also in the second trimester.

The Study of DNA Mutations of Phenylketonuria in Koreans

PURPOSE: Phenylketonuria(PKU) is an inborn error of metabolism and a genetic disorder resulting from a deficiency of phenylalanine hydroxylase(PAH) and decreased activity of tetrahydrobiopterin(BH4).In this study the correlation between the DNA mutation and clinical manifestations was investigated and PAH DNA mutations were compared bewteen Asian and Caucasian populations. METHODS: DNA was isolated from peripheral leukocytes. The PAH gene was amplified by Polymerase Chain Reaction(PCR) and the sequence was analyzed with Multiplex Ligation-dependent Probe Amplification(MLPA). RESULTS: We characterized the PAH gene of 102 independent Korean patients with PKU. PAH nucleotide sequence analysis revealed 44 different mutations, including 10 novel mutations comprising 9 missense mutations(N207D, K95del, A447P, G344D, P69S, S391I, A202T, G103S, and I306L) and 1 novel splice-site variant mutation(IVS10-3C>G). R243Q was the most prevalent mutation in this study. A259T has not previously been reported in Asian populations, but we found that this mutation had a frequency of 10.1% in our study. Furthermore, the genotypes of BH4 responsive patients were analyzed and were divided into two groups: BH4 medication-only group and BH4 medication with diet therapy group. In the BH4 medication-only group and BH4 medication with diet therapy group, R241C was the most common mutation. CONCLUSION: Novel mutations in the PAH gene of PKU patients are still being discovered. Additional information as to the frequency of mutations in the tetrahydrobiopterine responsive gene is also accumulating. We anticipate that knowledge of these PKU gene mutations will assist the diagnosis, genetic counseling, and therapeutic treatment of PKU patients in future.

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pah(enu2), by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

PHEX Gene Mutations and Genotype-Phenotype Analysis of Korean Patients with Hypophosphatemic Rickets

X-linked hypophosphatemic rickets (XLH) results from mutations in the PHEX gene. Mutational analysis of the PHEX gene in 15 unrelated Korean patients with hypophosphatemic rickets revealed eight mutations, including five novel mutations, in nine patients: two nonsense mutations, two missense mutations, one insertion, and three splicing acceptor/donor site mutations. Of these, c.64G>T, c.1699C>T, c.466_467 insAC, c.1174-1G>A, and c.1768+5G>A were novel mutations. To analyze the correlation between genotype and phenotype, phenotypes were compared between groups with and without a mutation, in terms of mutation location, mutation type, and sex. Skeletal disease tended to be more severe in the group with a mutation in the C-terminal half of the PHEX gene, but no genotype-phenotype correlation was detected in other comparisons. Further extensive studies of the PHEX gene mutations and analyses of the genotype-phenotype relationships are required to understand PHEX function and the pathogenesis of XLH.

Unilateral Parieto-Occipital Lobe Infarction Presenting with Optic Ataxia and Saccadic Abnormalties: A Case Report

Optic ataxia is characterized by an impaired visual control of the direction of arm reaching to a visual target, accompanied by defective hand orientation and grip formation. In humans, optic ataxia is associated with lesions of the superior parietal lobule, which also affect visually guided saccades and other forms of eye-hand coordination. A 67-year-old woman presented with sudden, unilateral, direct optic ataxia in the right homonymous half field and saccadic abnormalities which consisted of prolonged latency, undershoot dysmetria, and decreased velocity. Brain MRI showed a left parieto-occipital watershed infarction. It is suggested that lesions of the superior parietal lobule and the adjacent parietal eye field are responsible for optic ataxia and saccadic abnormalities, respectively.

Downregulation of neurotrophic factors in the brain of a mouse model of Gaucher disease: implications for neuronal loss in Gaucher disease

Gaucher disease is a glycosphingolipid storage disease caused by deficiency of glucocerebrosidase, resulting in the accumulation of glucosylceramide in lysosomes. The neuronopathic forms of this disease are associated with neuronal loss and neurodegeneration. However, the pathophysiological mechanisms leading to prenatal and neonatal death remain uncharacterized. To investigate brain dysfunction in Gaucher disease, we studied the effects of neurotrophic factors during development in a mouse model of Gaucher disease. The expression of brain-derived neurotrophic factor and nerve growth factor was reduced in the cerebral cortex, brainstem, and cerebellum of Gaucher mice, compared with that in wild-type mice. Extracellular signal-regulated kinase (ERK) 1/2 expression was downregulated in neurons from Gaucher mice and correlated with a decreased number of neurons. These results suggest that a reduction in neurotrophic factors could be involved in neuronal loss in Gaucher disease.

Recombinant adeno-associated virus mediated gene transfer in a mouse model for homocystinuria

Homocystinuria is a metabolic disorder caused by a deficiency of cystathionine b-synthase (CBS). The major clinical symptoms of this disease are mental retardation, lens dislocation, vascular disease with life-threatening thromboembolisms, and skeletal deformities. The major treatments for CBS deficiency include pharmacologic doses of pyridoxine or dietary restriction of methionine. There is currently no effective long-term treatment to lower the elevated plasma levels of homocysteine. However, gene therapy could be an effective novel approach for the treatment of homocystinuria. A recombinant adeno- associated virus vector carrying human CBS cDNA (rAAV-hCBS) was constructed and administered to CBS-/- mice by intramuscular (IM) and intraperitoneal (IP) injections. Serum homocysteine concentrations significantly decreased in treated mice compared with age-matched controls two weeks after treatment. The treated CBS-/- mice had life spans 3-7 days longer compared with untreated CBS-/- mice. In CBS-/- mice treated with rAAV-hCBS via IP injection, the vector was detected in all organs examined including liver, spleen, and kidney, and CBS gene expression was observed by immunohistochemical staining in the liver. These results indicate the efficacy of gene delivery and demonstrate the possibility of gene therapy mediated by AAV gene transfer in this mouse model of homocystinuria.

Upregulation of Proinflammatory Cytokines in the Fetal Brain of the Gaucher Mouse

Gaucher disease is caused by a deficiency of glucocerebrosidase. Patients with Gaucher disease are divided into three major phenotypes: chronic nonneuronopathic, acute neuronopathic, and chronic neuronopathic, based on symptoms of the nervous system, the severity of symptoms, and the age of disease onset. The characteristics of patients with acute neuronopathic- and chronic neuronopathic-type Gaucher disease include oculomotor abnormalities, bulbar signs, limb rigidity, seizures and occasional choreoathetoid movements, and neuronal loss. However, the mechanisms leading to the neurodegeneration of this disorder remain unknown. To investigate brain dysfunction in Gaucher disease, we studied the possible role of inflammation in neurodegeneration during development of Gaucher disease in a mouse model. Elevated levels of the proinflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and TNF-alpha, were detected in the fetal brains of Gaucher mice. Moreover, the levels of secreted nitric oxide and reactive oxygen species in the brains of Gaucher mice were higher than in wild-type mice. Thus, accumulated glucocerebroside or glucosylsphingosine, caused by glucocerebrosidase deficiency, may mediate brain inflammation in the Gaucher mouse via the elevation of proinflammatory cytokines, nitric oxide, and reactive oxygen species.

Episodic Ataxia Type 2 with Downbeating Nystagmus Caused by Mutation in the CACNA1A: A Case Report

Episodic ataxia type 2 (EA 2) is a rare disorder characterized by intermittent episodes of ataxia with interictal nystagmus. The authors report a patient with EA 2, who presented with recurrent episodes of vertigo, gait ataxia and interictal downbeat nystagmus, which had developed about 16 years before. The chromosomal analysis revealed a translocation between chromosome 7 and chromosome 19 (t(7;19)). The break point in chromosome 19 was the P13 locus of the CACNA1A gene.

Intra-articular Injection of IL-1beta Facilitated Formalin-induced Temporomandibular Joint Pain in Freely Moving Rats

The present study was performed to investigate the effects of intra-articular injection of interleukin-1beta (IL-1beta) on the formalin-induced temporomandibular joint (TMJ) pain. Under anesthesia, a 30-gauge needle was introduced into the right TMJ region for injection of formalin. Microinjection of 50 microliter of 5% formalin significantly produced noxious scratching behavioral response, and the scratching behavior lasted for 40 min. Although the responses produced by formalin injection were divided into two phases, the response of 1st phase did not significantly differ from the scratching behavior response in the saline-treated group. We examined the effects of intra-articular injection of IL-1beta on the number of noxious behavioral responses produced by 50microliter of 5% formalin injection. Intra-articular injection of 100 pg and 1 ng of IL-1beta significantly increased the number of behavioral responses of the 2nd phase, while 10 pg of IL-1beta did not change the formalin-induced behavioral responses. To investigate whether IL-1 receptor was involved in the intra-articular administration of IL-1beta-induced hyperalgesic response, IL-1 receptor antagonist (IL- ra, 50 ng) was administrated together with IL-1beta injection. IL-1beta receptor antagonist blocked IL-1beta- induced hyperalgesic response in the TMJ formalin test. These results suggest that intra-articular injection of IL-1beta facilitated the transmission of nociceptive information in the TMJ area.

Quantitative Analysis of SMN1 Gene and Estimation of SMN1 Deletion Carrier Frequency in Korean Population based on Real-Time PCR

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The delta delta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.