Preclinical Study on Biodistribution of Mesenchymal Stem Cells after Local Transplantation into the Brain

Therapeutic efficacy of mesenchymal stem cells (MSCs) is determined by biodistribution and engraftment in vivo.Compared to intravenous infusion, biodistribution of locally transplanted MSCs are partially understood. Here, we performed a pharmacokinetics (PK) study of MSCs after local transplantation. We grafted human MSCs into the brains of immune-compromised nude mice. Then we extracted genomic DNA from brains, lungs, and livers after transplantation over a month. Using quantitative polymerase chain reaction with human Alu-specific primers, we analyzed biodistribution of the transplanted cells. To evaluate the role of residual immune response in the brain, MSCs expressing a cytosine deaminase (MSCs/CD) were used to ablate resident immune cells at the injection site. The majority of the Alu signals mostly remained at the injection site and decreased over a week, finally becoming undetectable after one month. Negligible signals were transiently detected in the lung and liver during the first week. Suppression of Iba1-positive microglia in the vicinity of the injection site using MSCs/CD prolonged the presence of the Alu signals.After local transplantation in xenograft animal models, human MSCs remain predominantly near the injection site for limited time without disseminating to other organs. Transplantation of human MSCs can locally elicit an immune response in immune compromised animals, and suppressing resident immune cells can prolong the presence of transplanted cells. Our study provides valuable insights into the in vivo fate of locally transplanted stem cells and a local delivery is effective to achieve desired dosages for neurological diseases.

Cell Type-specific Knockout with Gli1-mediated Cre Recombination in the Developing Cerebellum

The inducible Cre-loxP system provides a useful tool for inducing the selective deletion of genes that are essential for proper development and enables the study of gene functions in properly developed animals. Here, we show that inducible Cre-loxP driven by the Gli1-promoter can induce cell-type-specific deletion of target genes in cerebellar cortical neurons. We used reporter mice containing the YFP (yellow fluorescence protein) gene at the Gt(ROSA)26Sor locus with a loxP -flanked transcriptional stop sequence, in which successful Cre-mediated excision of the stop sequence is indicated by YFP expression in Cre-expressing cells. Administration of tamoxifen during early postnatal days (P4~7) induces Cre-dependent excision of stop sequences and allows YFP expression in proliferating neuronal progenitor cells in the external granule layer and Bergmann glia in the Purkinje cell layer. A substantial number of YFP-positive progenitor cells in the external granule layer migrated to the internal granule cell layer and became granule cell neurons. By comparison, injection of tamoxifen during late postnatal days (P19~22) induces YFP expression only in Bergmann glia, and most granule cell neurons were devoid of YFP expression. The results indicate that the Gli1 promoter is temporarily active in progenitor cells in the external granule layer during the early postnatal period but constitutively active in Bergmann glia. We propose that the Gli1-mediated CreER system can be applied for the conditional deletion of genes of interest from cerebellar granule cell neurons and/or Bergmann glia.

Cell Type-specific Knockout with Gli1-mediated Cre Recombination in the Developing Cerebellum

The inducible Cre-loxP system provides a useful tool for inducing the selective deletion of genes that are essential for proper development and enables the study of gene functions in properly developed animals. Here, we show that inducible Cre-loxP driven by the Gli1-promoter can induce cell-type-specific deletion of target genes in cerebellar cortical neurons. We used reporter mice containing the YFP (yellow fluorescence protein) gene at the Gt(ROSA)26Sor locus with a loxP -flanked transcriptional stop sequence, in which successful Cre-mediated excision of the stop sequence is indicated by YFP expression in Cre-expressing cells. Administration of tamoxifen during early postnatal days (P4~7) induces Cre-dependent excision of stop sequences and allows YFP expression in proliferating neuronal progenitor cells in the external granule layer and Bergmann glia in the Purkinje cell layer. A substantial number of YFP-positive progenitor cells in the external granule layer migrated to the internal granule cell layer and became granule cell neurons. By comparison, injection of tamoxifen during late postnatal days (P19~22) induces YFP expression only in Bergmann glia, and most granule cell neurons were devoid of YFP expression. The results indicate that the Gli1 promoter is temporarily active in progenitor cells in the external granule layer during the early postnatal period but constitutively active in Bergmann glia. We propose that the Gli1-mediated CreER system can be applied for the conditional deletion of genes of interest from cerebellar granule cell neurons and/or Bergmann glia.

ERK Regulates NeuroD1-mediated Neurite Outgrowth via Proteasomal Degradation

Neurogenic differentiation 1 (NeuroD1) is a class B basic helix-loop-helix (bHLH) transcription factor and regulates differentiation and survival of neuronal and endocrine cells by means of several protein kinases, including extracellular signal-regulated kinase (ERK). However, the effect of phosphorylation on the functions of NeuroD1 by ERK has sparked controversy based on context-dependent differences across diverse species and cell types. Here, we evidenced that ERK-dependent phosphorylation controlled the stability of NeuroD1 and consequently, regulated proneural activity in neuronal cells. A null mutation at the ERK-dependent phosphorylation site, S274A, increased the half-life of NeuroD1 by blocking its ubiquitin-dependent proteasomal degradation. The S274A mutation did not interfere with either the nuclear translocation of NeuroD1 or its heterodimerization with E47, its ubiquitous partner and class A bHLH transcription factor. However, the S274A mutant increased transactivation of the E-box-mediated gene and neurite outgrowth in F11 neuroblastoma cells, compared to the wild-type NeuroD1. Transcriptome and Gene Ontology enrichment analyses indicated that genes involved in axonogenesis and dendrite development were downregulated in NeuroD1 knockout (KO) mice. Overexpression of the S274A mutant salvaged neurite outgrowth in NeuroD1-deficient mice, whereas neurite outgrowth was minimal with S274D, a phosphomimicking mutant. Our data indicated that a longer protein half-life enhanced the overall activity of NeuroD1 in stimulating downstream genes and neuronal differentiation. We propose that blocking ubiquitin-dependent proteasomal degradation may serve as a strategy to promote neuronal activity by stimulating the expression of neuron-specific genes in differentiating neurons.

Spatiotemporal Protein Atlas of Cell Death-Related Molecules in the Rat MCAO Stroke Model

Ischemic stroke and cerebral infarction triggered by the blockage of blood supply can cause damage to the brain via a complex series of pathological changes. Recently, diverse therapies have emerged as promising candidates for the treatment of stroke. These treatments exert therapeutic effects by acting on diverse target molecules and cells in different time windows from the acute to chronic phases. Here, using immunohistochemistry, we show pathophysiological changes in the brain microenvironment at the hyperacute (within 6 h), acute (1~3 days), subacute (7 days), and chronic (1 month) phases following ischemic injury. Ischemic injury in rats was induced by occluding the middle cerebral artery and was validated by magnetic resonance imaging. The progression of damage to the brain was evaluated by immunohistochemistry for NeuN⁺ neurons, GFAP⁺ astrocytes, and Iba1⁺ microglia, and by the emergence of the cell death-related molecules such as AIF, FAF1, and activated caspase-3. Our data regarding the spatial and temporal information on pathophysiological changes may warrant the investigation of the timing of administration of therapeutic treatments in preclinical studies with an animal model of stroke.

Erratum: Analysis of Dosage Mutation in PARK2 among Korean Patients with Early-Onset or Familial Parkinson's Disease

Authors would like to change annotation of variants in Table 1 and its legend according to HGVS nomenclature recommendation.

Analysis of Dosage Mutation in PARK2 among Korean Patients with Early-Onset or Familial Parkinson's Disease

BACKGROUND AND PURPOSE: There is some controversy regarding heterozygous mutations of the gene encoding parkin (PARK2) as risk factors for Parkinson's disease (PD), and all previous studies have been performed in non-Asian populations. Dosage mutation of PARK2, rather than a point mutation or small insertion/deletion mutation, was reported to be a risk factor for familial PD; dosage mutation of PARK2 is common in Asian populations. METHODS: We performed a gene-dosage analysis of PARK2 using real-time polymerase chain reaction for 189 patients with early-onset PD or familial PD, and 191 control individuals. In the case of PD patients with heterozygous gene-dosage mutation, we performed a sequencing analysis to exclude compound heterozygous mutations. The association between heterozygous mutation of PARK2 and PD was tested. RESULTS: We identified 22 PD patients with PARK2 mutations (11.6%). Five patients (2.6%) had compound heterozygous mutations, and 13 patients (6.9%) had a heterozygous mutation. The phase could not be determined in one patient. Three small sequence variations were found in 30 mutated alleles (10.0%). Gene-dosage mutation accounted for 90% of all of the mutations found. The frequency of a heterozygous PARK2 gene-dosage mutation was higher in PD patients than in the controls. CONCLUSIONS: Heterozygous gene-dosage mutation of PARK2 is a genetic risk factor for patients with early-onset or familial PD in Koreans.

Simultaneous deletion of floxed genes mediated by CaMKIIalpha-Cre in the brain and in male germ cells: application to conditional and conventional disruption of Goalpha

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIalpha) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIalpha-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice generated by crossing CaMKIIalpha-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIalpha-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Delta)) without inheriting the Cre transgene. The Gnao(Delta/Delta) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIalpha-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIalpha-Cre mice as breeding pairs.

Differences in Clinical Manifestations between Bipolar I and Bipolar II Disorders in Korean Population / 신경정신의학

OBJECTIVES : Whether bipolar II disorder (BP-II) is simply a milder form of bipolar I disorder (BP-I) or a valid diagnostic category that could be separated from BP-I, is an issue still under consideration. Investigations exploring differential clinical and biological features of the two conditions are needed to resolve the controversies. This study aimed to obtain a comprehensive view of differences in clinical course and symptoms characteristics between BP-I and BP-II. METHODS : 44 BP-I and 26 BP-II patients were assessed using the Diagnostic Interview for Genetic Studies (DIGS), Korean version. Demographic data, age at onset, number of (hypo) manic/ depressive episodes, the duration of illness, polarity at onset, seasonality, rapid cycling, atypical depression and symptom profiles of each episode were evaluated. RESULTS : BP-II patients experienced depressive episodes more frequently than BP-I patients after illness onset (U=240.5, p=0.008). More BP-II patients showed seasonality (34.9% vs. 61.5%) and a rapid cycling course (4.5% vs. 18.2%). When comparing symptom profiles of manic/hypomanic episodes, irritable mood, decreased sleep need, inattention, reckless behavior, arrogant/provocative attitude and frequent outbursts of anger were less encountered in BP-II patients. In depressive episodes, leaden paralysis and psychomotor agitation were more frequently observed in BP-II patients. There was no significant difference between the two groups in psychotic symptoms of depressive episode. CONCLUSION : BP-I and BP-II disorders showed differences in clinical courses and symptom profiles. BP-II disorder seems to be less severe than BP-I disorder with regard to the intensity of manic symptoms, but more severe with respect to frequencies of depressive episodes. These results provide additional evidence supporting the distinction of BP-I and BP-II as separate diagnos-tic categories that might have different genetic profiles and/or biological mechanisms.

Differences in Clinical Manifestations between Bipolar I and Bipolar II Disorders in Korean Population / 신경정신의학

OBJECTIVES : Whether bipolar II disorder (BP-II) is simply a milder form of bipolar I disorder (BP-I) or a valid diagnostic category that could be separated from BP-I, is an issue still under consideration. Investigations exploring differential clinical and biological features of the two conditions are needed to resolve the controversies. This study aimed to obtain a comprehensive view of differences in clinical course and symptoms characteristics between BP-I and BP-II. METHODS : 44 BP-I and 26 BP-II patients were assessed using the Diagnostic Interview for Genetic Studies (DIGS), Korean version. Demographic data, age at onset, number of (hypo) manic/ depressive episodes, the duration of illness, polarity at onset, seasonality, rapid cycling, atypical depression and symptom profiles of each episode were evaluated. RESULTS : BP-II patients experienced depressive episodes more frequently than BP-I patients after illness onset (U=240.5, p=0.008). More BP-II patients showed seasonality (34.9% vs. 61.5%) and a rapid cycling course (4.5% vs. 18.2%). When comparing symptom profiles of manic/hypomanic episodes, irritable mood, decreased sleep need, inattention, reckless behavior, arrogant/provocative attitude and frequent outbursts of anger were less encountered in BP-II patients. In depressive episodes, leaden paralysis and psychomotor agitation were more frequently observed in BP-II patients. There was no significant difference between the two groups in psychotic symptoms of depressive episode. CONCLUSION : BP-I and BP-II disorders showed differences in clinical courses and symptom profiles. BP-II disorder seems to be less severe than BP-I disorder with regard to the intensity of manic symptoms, but more severe with respect to frequencies of depressive episodes. These results provide additional evidence supporting the distinction of BP-I and BP-II as separate diagnos-tic categories that might have different genetic profiles and/or biological mechanisms.

A Study on the Nutrition Knowledge and Nutritional Status of Food and Nutrition Major and Physical Science Major Female Students / 대한지역사회영양학회지

The purpose of this study is to examine the nutrition knowledge, characteristics related to, nutrient intakes, anthropometrics, biochemical indices of university female students by major (food and nutrition versus physical science). Data were taken from 120 university female students (60 from each major) and the results follows. The mean age of the subjects was 19.6 years old. Most of subjects responded that they were healthy; subjects majoring in physical science scored higher in regular exercise (p < 0.01) and were more satisfied with their own body figures (p < 0.01), compared with the counterparts. The total nutrition knowledge score was 81.01 +/- 12.3 for food and nutrition majors and 72.5 +/- 15.2 points for physical science majors. (p < 0.01) The percentages of body fat were significantly higher in the food and nutrition students than the counterparts. (p < 0.001) The result of biochemical analysis showed that both groups were in normal range. But there was some statistically significant difference between groups in GTP, HDL-cholesterol and ALP levels. It suggests that regular exercise might have a positive effect on the body. This study showed that although both groups had different knowledge of nutrition, there was not much difference in the intakes of nutrients. But especially, the intakes of calcium and iron were quite low in both groups. Subjects majoring in Physical science had more lipid intake, but they had lower body fat. This suggests that regular exercise in this group might have effects on the percentages of body fat. In future study, nutrition education might be planned to increase nutrition knowledge and to connect nutrition knowledge to eating behaviors and to promote health to regular exercise. Also, the desire and the social perception for pursuing alean body figure and being underweight should be changed for optimal health

Trinucleotide Repeats Number in SCA2, SCA3, and SCA17 in Early-Onset Parkinson's Disease

BACKGROUND: Abnormal expansion of trinucleotide repeats in genes causing spinocerebellar ataxias such as SCA2, SCA3, SCA8, or SCA17 was reported in sporadic or familial Parkinson's disease. Genetic factors play an important role especially in early-onset Parkinson's disease (EOPD). To investigate mutations of ATXN2, ATXN3, and TBP as a possible cause in Korean EOPD, we analyzed mutations in these genes. We also investgated the possibility that trinucleotide repeats numbers in these genes contribute to the development of EOPD. METHODS: Mutation analysis of ATXN2, ATXN3, and TBP was done in 153 EOPD defined as age-at-onset before 51. Distribution of CAG repeats numbers were compared between EOPD and age- and sex-matched controls. RESULTS: No patients with EOPD had CAG repeats numbers in ATXN2, ATXN3, and TBP in mutation range. There was no difference in the distribution of CAG repeats between EOPD and controls, although we found a trend that CAG repeats numbers in ATXN3 appear larger in EOPD than in controls. CONCLUSIONS: Mutations of genes causing SCA2, SCA3, or SCA17 may not be a common genetic cause in Korean EOPD.

Utility of polymerase chain reaction(PCR) according to sampling time in CSF and stool specimens from patient with aseptic meningitis / 소아과

PURPOSE: Enteroviruses are the most common cause of aseptic meningitis in patients of all ages. A definite diagnosis of enteroviral meningitis can be established by detection of virus directly in CSF specimens. But this is time-consuming and lacks sensitivity, so polymerase chain reaction(PCR) detecting of viral RNA in patient specimens such as CSF, stool has been demonstrated. But little is known about the influence of sampling time on the results of CSF PCR and stool PCR. We investigated diagnostic utility of PCR of CSF and stool according to sampling time after the onset of symptoms. METHODS: PCR results were analyzed according to sampling time for 42 patients diagnosed aseptic meningits in our hospital from 11(th) January to 30(th) August, 2005. RESULTS: The diagnostic yield of the test was higher of CSF specimens obtained 2 days after onset(positive PCR results 1/24, 4.2 percent)(P=0.001). Instead, positive PCR results of fecal specimens maintained highly(average 90.5 percent), 10 cases had also positive PCR results even 5-6 days after onset. 10 cases of CSF specimens had positive enterovirus PCR results containing coxsackievirus B5 (n=6), coxsackievirus B3(n=3). 38 cases of stool specimens had positive enterovirus PCR results containing echovirus 18(n=7), echovirus 9(n=3), coxsackievirus B5(n=8), coxsackievirus B3(n=3). 6 cases(coxackie B5) had positive CSF PCR and stool PCR, both. CONCLUSION: Stool PCR was clinically sensitive for detecting enterovirus during enteroviral meningits and could give a presumptive diagnosis throughout the disease course. A definite diagnosis was obtained by CSF PCR, but its utility was clearly lower for samples obtained >2 days after clinical onset. Therefore, it is recommended that, in addition to performance of CSF PCR, fecal samples obtained from patients with suspected enteroviral meningitis should be tested by PCR, especially when the duration of symptoms is >2 days.

A Case of Jarcho-Levin Syndrome with Intrathoracic Kidney / 소아과

In 1938, Jarcho and Levin initially described shortening of the trunk, prominent occiput, broad forehead, multiple vertebral defects and ribs anomaly, short neck, increased anteroposterior chest diameter, lordosis, kyphoscoliosis. After that, Jarcho-Levin syndrome is an eponym that has been used to describe a variety of clinical phenotypes. We examined a girl who was suspected as suffering from Jarcho-Levin syndrome because she had shortening of the trunk, multiple vertebral defects and ribs anomaly, short neck, increased anteroposterior chest diameter, lordosis, kyphoscoliosis. We report a case of Jarcho-Levin syndrome with intrathoracic kidney, and review related literature.

Effect of Intranasal Corticosteroid Therapy for Perennial Nonallergic Rhinitis and Perennial Allergic Rhinitis / 소아알레르기및호흡기학회지

PURPOSE: The efficacy of intranasal corticosteroids for the treatment of allergic rhinitis has been reported. But the efficacy of intranasal corticosteroids has not been compared between perennial nonallergic rhinitis (PNAR) and perennial allergic rhinitis (PAR). METHODS: Twenty-three patients with PNAR and 19 patients with PAR were enrolled in this study. Every patient received fluticasone propionate (FP) 200 g (100 g, bid) daily for 4weeks. Control group (n=16) was received only anti-histamine (hydroxyzine 0.6 mg/kg/dose) intermittently. Efficacy of FP was evaluated by the mean change in nasal congestion, rhinorrhea, nasal itching, sneezing and total nasal symptom score (a sum of patient ratings of nasal congestion, rhinorrhea, nasal itching, and sneezing). RESULTS: Both groups (PNAR and PAR) showed similar improvement of nasal symptom with FP 200 g compared with control. (P< 0.05) In the total population, both groups showed significant improvements from baseline in TNSS compared with control during each week of treatment. (P< 0.05) In PNAR and PAR, nasal congestion was significantly improved more than in control. (P< 0.01, P< 0.01) Rhinorrhea and itching in PAR were improved more than in control. (P=0.02, P=0.03) In sneezing, all three groups showed no differences. (P=1.00, P=0.31, P= 0.29) CONCLUSION: Intranasal FP is an effective treatment for perennial nonallergic rhinitis as same as perennial allergic rhinitis.