Kallmann syndrome (KS) is a rare pediatric disease with major manifestations of olfactory dysfunction and hypogonadotropic hypogonadism. Five children (4 boys and 1 girl) with KS reported in this article were aged between 6 months and 19 years at the time when they attended the hospital. All the children had the clinical manifestation of hypogonadotropic hypogonadism; in addition, three children had olfactory dysfunction (two were found to have olfactory bulb dysplasia on magnetic resonance imaging), one had cleft lip and palate, and one had micropenis and cryptorchidism with right renal agenesis during infancy. All the five children had normal karyotype and their parents had normal clinical phenotypes. The uncle of one child had underdeveloped secondary sexual characteristics and olfactory disorder since childhood. High-throughput sequencing found two known heterozygous missense mutations in the FGFR1 gene, i.e., c.1097C>T(p.P366L) and c.809G>C(p.G270A), in two children. One child had a novel frameshift mutation, c.1877_1887/p.S627Tfs*6, in the KAL1 gene; this deletion mutation caused a frameshift in base sequence and produced truncated proteins, which led to a significant change in protein structure, and thus it was highly pathogenic. It is concluded that KS has great clinical and genetic heterogeneity and can be accompanied by incomplete dominant inheritance and that gene detection helps with the diagnosis of this disease.
Adolescent ; Child ; Child, Preschool ; DNA Mutational Analysis ; Extracellular Matrix Proteins ; Female ; Heterozygote ; Humans ; Hypogonadism ; Infant ; Kallmann Syndrome ; Male ; Mutation ; Nerve Tissue Proteins ; Receptor, Fibroblast Growth Factor, Type 1 ; Young Adult

Mesenchymal stem cell (MSC)-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations: CD51/CD140α, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells (DMSCs) from heterogeneous periodontal ligament cells (PDLCs). Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51(+)/CD140α(+), 0.8% were CD271(+), and 2.4% were STRO-1(+)/CD146(+). Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271(+) DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.
Adaptor Proteins, Signal Transducing ; analysis ; Adult ; Aggrecans ; analysis ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; CD146 Antigen ; analysis ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Collagen Type II ; analysis ; Core Binding Factor Alpha 1 Subunit ; analysis ; Flow Cytometry ; methods ; Homeodomain Proteins ; analysis ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; physiology ; Multipotent Stem Cells ; cytology ; physiology ; Nerve Tissue Proteins ; analysis ; Osteogenesis ; physiology ; Periodontal Ligament ; cytology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis ; SOX9 Transcription Factor ; analysis ; Time Factors ; Transcription Factors ; analysis

Mesenchymal stem cells (MSCs) are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow (BMSCs), adult MSCs are isolated from craniofacial tissues including dental pulp tissues (DPs) using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cells (DMSCs). In this study, we used different combinations of surface markers (CD51/CD140α, CD271, and STRO-1/CD146) to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells (DPCs) obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51(+)/CD140α(+), 10.6% were CD271(+), and 0.3% were STRO-1(+)/CD146(+). Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271(+) DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271(+) DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.
Adult ; Adult Stem Cells ; cytology ; Antigens, CD ; analysis ; Antigens, Surface ; analysis ; Biomarkers ; analysis ; CD146 Antigen ; analysis ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Lineage ; Cell Separation ; methods ; Cells, Cultured ; Chondrogenesis ; physiology ; Dental Pulp ; cytology ; Flow Cytometry ; methods ; Humans ; Integrin alphaV ; analysis ; Mesenchymal Stromal Cells ; cytology ; Multipotent Stem Cells ; cytology ; Nerve Tissue Proteins ; analysis ; Odontogenesis ; physiology ; Receptor, Platelet-Derived Growth Factor alpha ; analysis ; Receptors, Nerve Growth Factor ; analysis

OBJECTIVETo summarize the clinical features of idiopathic hypogonadotropic hypogonadism (IHH) diagnosed during childhood, and detect mutations in KAL1 and FGFR1, acting as key clues for diagnoses.

METHODWe collected and analyzed clinical data of 21 cases (including demographic data, chief complaint, history of present illness, family history, physical examination, laboratory tests and imaging studies, etc.) diagnosed with IHH from December 2008 to February 2013. Polymerase chain reaction and gene sequencing was applied to detect mutations on KAL1 and FGFR1. Fifty healthy unrelated individuals were choosen as controls.

RESULTOf 21 patients with IHH, 19 were males and 2 females, they visited us initially from 8-17 years old, with an average of (13.58 ± 2.38) years old. Sixteen cases were KS patients (76%). One boy reported abnormal sense of smelling but having olfactory perfect picture on MRI; 2/19 male cases had no puberty when they were over 13-14 years old without abnormal external genitalia. 8/19 cases only had small penis, 8/19 had both of cryptorchidism and small penis, and the Case 2 also had hypospadias. One boy had cryptorchidism combined with a normal penis. Only 2 girls diagnosed as IHH who visited us because of no puberty signs when they were 13 and 16 years old, respectively. Other clinical manifestations included: one with gynecomastia, 2 had mental retardation, and one was deaf; one with high palatal arch; one with mirror-movement and one with left renal agenesis but normal renal function respectively. Laboratory tests showed that the basic testosterone (T) is low and with inappropriately low or normal gonadotropin hormones. The results of cases of standard human chorionic gonadotropin (HCG) test of 7 cases out of 19 male children's were normal (testosterone>1 100 ng/L), and another nine cases continued to complete the extended HCG test, and the testosterone levels of two of them (cases 6, 8) were still lower than 1 000 ng/L. Family history: the parents in 9/21 family had delayed puberty, involving only one parent in 6 families, involving both in 2 families and the other one was an uncle having micropenis with a child. Among these 21 cases, only one boy's father had hyposmia and his first emission age was 14-15 years. Eleven patients accompanied abnormal sense of smelling and the olfactory organ abnormalities on MRI, 4 had olfactory organ abnormalities on MRI while they had good smelling function self-reportedly. We got 15 samples (12 KS and 3 nIHH cases) to screen the mutation of KAL1 (14 exons) and FGFR1 (18 exons). A splicing mutation c.1062+1G>A in KAL1 is identified in case 17 with IHH. One novel heterozygous FGFR1 mutation, a single base deletion mutation on the exon 1 c.27delC is identified in case 14. This mutation causes the premature termination codons.

CONCLUSIONThis pilot research showed that IHH/KS diagnosis in children depends on clinical manifestation rather than gene analysis. Small penis or cryptorchidism, smelling abnormality and positive familial history may contribute to the KS/HH diagnosis. MRI of olfactory bulb acts as important proof for diagnosis of KS. Mutations in KAL1 and FGFR1 gene are not main causes of Kallmann syndrome.

Adolescent ; Child ; DNA Mutational Analysis ; Exons ; genetics ; Extracellular Matrix Proteins ; genetics ; Female ; Heterozygote ; Humans ; Hypogonadism ; diagnosis ; genetics ; Kallmann Syndrome ; genetics ; Male ; Mutation ; genetics ; Nerve Tissue Proteins ; genetics ; Olfaction Disorders ; Receptor, Fibroblast Growth Factor, Type 1 ; genetics ; Sexual Maturation

OBJECTIVETo observe the mRNA expression of p75NTR (p75 neurotrophin receptor) and the amount of neuronal cells apoptosis in lumbar-sacral spinal cord at different time points after the acute cauda equina compression in rats and to explore their correlation.

METHODSSixty adult female Sprague Dawley(SD) rats were randomly divided into the normal control group and the compression groups. The acute cauda equine compression model was established as placing a silicon gel rubber at L(3,4) level of the vertebral canal which represented about 70% to 80% compression to the cross section. The whole L(1,2) level of spinal cords were harvested at 1, 3, 5, 7, 14, 28 d after operation in compression group. Tunel method was applied to observe cell apoptosis and RT-PCR was used to detect the p75NTR mRNA expression. SPSS 13.0 statistical software was adopted to help analysis.

RESULTSIn the compression group, both the nerve cells apoptosis and the p75 mRNA expression existed the trend of low-high-low synchronally compared with the control group, there was a significant difference (P < 0.05) among comprssion groups at different time points,there was a significant difference in changes (P < 0.05). p75NTR of mRNA expression and lumbosacral nerve cells apoptosis was in a positive correlation.

CONCLUSIONAfter acute cauda equina compression, p75NTR mRNA expression is closely related to the neuronal apoptosis, which plays an important role in the molecular mechanism of the CES.

Animals ; Apoptosis ; Cauda Equina ; Disease Models, Animal ; Female ; Polyradiculopathy ; etiology ; metabolism ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Nerve Growth Factor ; genetics ; Spinal Cord Compression ; metabolism

OBJECTIVETo study the mechanism of brain development delay in rats with intrauterine growth retardation (IUGR) by examining the expression of nerve growth factor (NGF) and tyrosine kinase receptor A (Trk A) in the brain.

METHODSThirty-two pregnant rats were randomly fed with a normal diet (control group) or lower protein diet (IUGR group) during pregnancy (n=16 each). The pup rats were sacrificed at 0, 7, 14 or 21 days after birth. The protein levels of NGF and TrkA in the brain were determined by Western blot and immunohistochemistry staining.

RESULTSThe levels of NGF and TrkA in the brain in pup rats of the IUGR group were significantly lower than those in the control group 0, 7, 14 and 21 days after birth.

CONCLUSIONSThe decreased expression of protein levels of NGF and TrkA in the brain might be one of the causes of brain development delay in IUGR rats.

Animals ; Birth Weight ; Brain Chemistry ; Female ; Fetal Death ; epidemiology ; Fetal Growth Retardation ; epidemiology ; metabolism ; Immunohistochemistry ; Nerve Growth Factor ; analysis ; Pregnancy ; Rats ; Rats, Wistar ; Receptor, trkA ; analysis

OBJECTIVETo investigate the roles of FIZZ1 and NOTCH1 in the pathogenesis of asthma and the effect of rosiglitazone on airway remodeling.

METHODSForty-five healthy 6 to 8-week-old Sprague-Dawley rats were randomly divided into a control group and asthma groups with and without rosiglitazone treatment. The paraffin slices of lung tissues were made to assess the histological changes. a-SMA protein, a specific marker of airway remodeling, in lung tissues was measured by immunohistochemistry. FIZZl-mRNA and NOTCH1-mRNA expression in lung tissues was measured by RT-PCR.

RESULTSThe characteristic changes of airway remodeling were observed in the untreated asthma group. The histological changes in the airway were less severe in the rosiglitazone treated asthma group. Positive a-SMA staining, FIZZl-mRNA and NOTCH1-mRNA were highly expressed in peribronchial lung sections isolated from the untreated asthma group. Rosiglitazone treatment decreased significantly the expression of a-SMA protein, FIZZl-mRNA and NOTCH1-mRNA compared with the untreated asthma group, but the expression of a-SMA protein, FIZZl-mRNA and NOTCH1-mRNA in the rosiglitazone treated asthma group remained higher than the control group. a-SMA expression was positively correlated with FIZZl-mRNA (r=0.826, P<0.01) and NOTCH1-mRNA expression (r=0.9, P<0.01). FIZZl-mRNA expression was positively correlated with NOTCH1-mRNA expression (r=0.76, P<0.01).

CONCLUSIONSFIZZl and NOTCH1 may induce an increase in a-SMA expression. FIZZl and NOTCH1 play a critical role in the process of airway remodeling. Rosiglitazone treatment may inhibit airway remodeling in asthmatic rats.

Actins ; Airway Remodeling ; Animals ; Asthma ; etiology ; pathology ; Lung ; metabolism ; pathology ; Male ; Nerve Growth Factor ; genetics ; physiology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; physiology

BACKGROUND AND OBJECTIVES: We investigated the expression of glucose uptake transporter-1 (GLUT-1) and epidermal growth factor receptor (EGFR) in the head and neck squamous cell cancer (HNSCC) and their influence on survival rate. We also aimed to demonstrate the relationships between the expressions of GLUT-1 and EGFR and the maximal standardized uptake values (mSUV) of fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET). SUBJECTS AND METHOD: Surgical specimens from 50 patients with HNSCC, who had undergone surgical intervention, were first processed for tissue array and then for immunochemistry for GLUT-1 and EGFR. Expressions of GLUT-1 and EGFR were analyzed according to the differentiation of tumor, T & N stage and invasiveness of the tumor etc. The relationship between PET-mSUV and the expressions of EGFR and GLUT-1 was evaluated as well as its possibility as a prognostic factor of SUV. RESULTS: The expression of EGFR was significantly high in the lymph node of the positive group. Associated with the expression of GLUT-1, a tendency of increased index score (IS) values in the advanced T stages was observed but it was not statistically significant. The mSUV in FDG-PET showed statistically significant association with the maximal tumor size (pearson correlation 0.569, p=0.023); however, no association with GLUT-1 and EGFR was observed. A universal strong expression of GLUT-1 could have made it difficult to figure out statistically significant correlations. Disease free survival was to be influenced by T stage, moderate and poor differentiation and GLUT-1 expression. Only the T stages had independent significance in the multivariate analysis using the Cox regression model. CONCLUSION: We found that the expression of GLUT-1 could not show statistically significant correlation with PET-mSUV. Since the expression of EGFR has significant correlation with lymph node metastasis, EGFR may have the possibility of being a target for anticancer molecular therapy.
Collodion ; Disease-Free Survival ; Glucose ; Head ; Humans ; Immunochemistry ; Lymph Nodes ; Multivariate Analysis ; Neck ; Neoplasm Metastasis ; Neoplasms, Squamous Cell ; Positron-Emission Tomography ; Receptor, Epidermal Growth Factor ; Recurrent Laryngeal Nerve ; Survival Rate ; Thyroid Gland

OBJECTIVERecent studies have indicated that the signal pathway of NgR-P75NTR- RhoA plays a key role in nerve injury and remodeling, but its exact mechanism and the role of the downstream molecule RhoA regulated by P75NTR remain unclear in hypoxia-ischemia (HI) neonatal animals. The present study was designed to assess the expression of P75NTR protein and RhoA mRNA in neonatal white matter and to investigate their relationship in newborn rats with white matter damage (WMD).

METHODSThe rat WMD model was established by the ligation of right common carotid artery, followed by 6% hypoxia exposure for 4 hrs. The control group was sham-operated, without HI treatment. The histological changes of brain tissue were observed under light and electron microscopes. Expression of P75NTR protein and RhoA mRNA in the brain white matter after 12, 24, 48 and 72 hrs and 7 days of HI were detected by RT-PCR and immunohistochemistry, respectively.

RESULTSPeriventricular white matter damage was observed by 48 hrs of HI. Expression of P75NTR protein increased in the striatum and callosum zones at 12 hrs, peaked at 48 hrs, and remained at a higher level than control until 72 hrs of HI in the WMD group (P < 0.01). After 7 days of HI expression of P75NTR protein was no longer statistically different from controls. The RhoA mRNA was higher in the WMD group for the first 72 hrs and then declined to control values.

CONCLUSIONSIncreased P75NTR protein might mediate apoptosis of nerve cells and inhibit the regeneration of neuron axons. The subsequent decline back to control value may be correlated with the aggregation of necrosis of nerve cells after HI. The patterns of RhoA mRNA expression were consistent with those of P75NTR protein, suggesting that the increased P75NTR level may promote RhoA mRNA expression.

Animals ; Animals, Newborn ; Brain ; pathology ; ultrastructure ; Female ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; Immunohistochemistry ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptor, Nerve Growth Factor ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; rhoA GTP-Binding Protein ; genetics

OBJECTIVETo investigate the expression and pattern of vascular endothelial growth factor (VEGF) and its fetal liver kinase-1 (Flk-1) receptor in spinal cord and dorsal root ganglia after neurotomy of sciatic nerve in rats.

METHODSForty-five adult male Wistar rats were divided randomly into a control group (n=5) and an experimental group (n=40). The bilateral sciatic nerves of the rats in the experimental group underwent neurotomy and the L4-L6 spinal cord and the corresponding dorsal root ganglia were harvested respectively at 8 hours, and 1, 3, 5, 7, 10, 14 and 21 days (8 subgroups with 5 rats each) after operation. The rats in the control group only underwent an exposure of sciatic nerve without neurotomy. Immunohistochemistry and image analysis were used to study the expression of VEGF and its Flk-1 receptor.

RESULTSBoth VEGF and Flk-1 receptor expressed in the normal rat spinal cord and dorsal root ganglia. In response to neurotomy, their expression reached a higher level and persisted for a short time then declined to the normal level rapidly. Besides, positive staining of Flk-1 was observed in both glial cells and nerve fibers, which located in the white matter of the spinal cord.

CONCLUSIONSVEGF can promote the regeneration of peripheral nerves from the angle of central neurons, which establishes the experimental and theoretical foundation for VEGF treating peripheral nerve injuries.

Analysis of Variance ; Animals ; Ganglia, Spinal ; metabolism ; Immunoenzyme Techniques ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Sciatic Nerve ; metabolism ; surgery ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; Vascular Endothelial Growth Factors ; biosynthesis