ABSTRACT
BACKGROUND AND AIM: Metabolic syndromes are prevalent worldwide and result in various complications including obesity, cardiovascular disease and type II diabetes. Betulinic acid (BA) is a naturally occurring triterpenoid that has anti-inflammatory properties. We hypothesized that treatment with BA may result in decreased body weight gain, adiposity and hepatic steatosis in a diet-induced mouse model of obesity. METHODS AND RESULTS: Mice fed a high-fat diet and treated with BA showed less weight gain and tissue adiposity without any change in calorie intake. Gene expression profiling of mouse tissues and cell lines revealed that BA treatment increased expression of lipid oxidative genes and decreased that of lipogenesis-related genes. This modulation was mediated by increased AMP-activated protein kinase (AMPK) phosphorylation, which facilitates energy expenditure, lipid oxidation and thermogenic capacity and exerts protective effects against obesity and nonalcoholic fatty liver disease. Overall, BA markedly inhibited the development of obesity and nonalcoholic fatty liver disease in mice fed a high-fat diet, and AMPK activation in various tissues and enhanced thermogenesis are two possible mechanisms underlying the antiobesity and antisteatogenic effects of BA. CONCLUSIONS: The current findings suggest that treatment with BA is a potential dietary strategy for preventing obesity and nonalcoholic fatty liver disease.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Adipocytes/drug effects , Anti-Obesity Agents/pharmacology , Energy Metabolism/drug effects , Liver/drug effects , Non-alcoholic Fatty Liver Disease/prevention & control , Obesity/prevention & control , Triterpenes/pharmacology , 3T3-L1 Cells , Adipocytes/enzymology , Adipocytes/pathology , Adiposity/drug effects , Animals , Diet, High-Fat , Disease Models, Animal , Enzyme Activation , Liver/enzymology , Liver/pathology , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/enzymology , Non-alcoholic Fatty Liver Disease/pathology , Non-alcoholic Fatty Liver Disease/physiopathology , Obesity/enzymology , Obesity/pathology , Obesity/physiopathology , Pentacyclic Triterpenes , Phosphorylation , Signal Transduction , Weight Gain/drug effects , Betulinic AcidABSTRACT
OBJECTIVE: Studies have indicated that weight regain following weight loss predisposes obese individuals to metabolic disorders; however, the molecular mechanism of this potential adverse effect of weight regain is not fully understood. Here we investigated global transcriptome changes and the immune response in mouse white adipose tissue caused by weight regain. DESIGN: We established a diet switch protocol to compare the effects of weight regain with those of weight gain without precedent weight loss, weight loss maintenance and chow diet. We conducted a time course analysis of global transcriptome changes in gonadal white adipose tissue (gWAT) during the weight fluctuation. Co-expression network analysis was used to identify functional modules associated with the weigh regain phenotype. Immune cell populations in gWAT were characterized by flow-cytometric immunophenotyping. Metabolic phenotypes were monitored by histological analysis of adipose tissue and liver, and blood-chemistry and body weight/composition analyses. RESULTS: In total, 952 genes were differentially expressed in the gWAT in the weight regain vs the weight gain group. Upregulated genes were associated with immune response and leukocyte activation. Co-expression network analysis showed that genes involved in major histocompatibility complex I and II-mediated antigen presentation and T-cell activation function were upregulated. Consistent with the transcriptome analysis results, flow cytometry demonstrated significant increases in subsets of T cells and proinflammatory M1 macrophages in the gWAT in the weight regain as compared to the weight gain group. In addition, upregulation of adaptive immune responses was associated with high incidence of adipocyte death and upregulation of high mobility group box 1, a well-known component of damage-associated molecular patterns. CONCLUSIONS: Our global transcriptome analysis identified weight regain-induced activation of adaptive immune responses in mouse white adipose tissue. Results suggest that activation of adipocyte death-associated adaptive immunity in adipose tissue may contribute to unfavorable metabolic effects of weight regain following weight loss.
Subject(s)
Adipose Tissue, White/immunology , Adipose Tissue, White/metabolism , Transcriptome/physiology , Weight Gain/immunology , Weight Gain/physiology , Adipose Tissue, White/chemistry , Animals , Gene Expression Profiling , Gonads/chemistry , Gonads/metabolism , Liver/chemistry , Liver/metabolism , Male , Mice , Mice, Inbred C57BLABSTRACT
Resonant soft-x-ray scattering measurements have been performed to investigate interface electronic structures of (LaAlO(3)/SrTiO(3)) superlattices. Resonant scattering intensities at superlattice reflections show clear evidence of degeneracy lifting in t(2g) states of interface Ti ions. Polarization dependence of intensities indicates the energy of d(xy) states is lower by ~1 eV than two other t(2g) states. The energy splitting is insensitive to epitaxial strain. The orbital reconstruction is induced by oxygen vacancies and confined to the interface within two unit cells, indicating charge compensation at the polar interfaces.
Subject(s)
Models, Chemical , Oxides/chemistry , Oxygen/chemistry , Strontium/chemistry , Titanium/chemistry , X-Ray Diffraction/methods , Aluminum/chemistry , Cations/chemistry , Lanthanum/chemistryABSTRACT
AIMS: Previously, we selected a bacterial strain (GSE09) antagonistic to Phytophthora capsici on pepper, which produced a volatile compound (2,4-di-tert-butylphenol), inhibiting the pathogen. In this study, we identified strain GSE09 and characterized some of the biological traits of this strain in relation to its antagonistic properties against P. capsici. In addition, we examined bacterial colonization on the root surface or in rhizosphere soil and the effect of various concentrations of the volatile compound and strain GSE09 on pathogen development and radicle infection as well as radicle growth. METHODS AND RESULTS: Strain GSE09 was identified as Flavobacterium johnsoniae, which forms biofilms and produces indolic compounds and biosurfactant but not hydrogen cyanide (HCN) with little or low levels of antifungal activity and swimming and swarming activities. Fl. johnsoniae GSE09 effectively colonized on pepper root, rhizosphere, and bulk (pot) soil, which reduced the pathogen colonization in the roots and disease severity in the plants. Various concentrations of 2,4-di-tert-butylphenol or strain GSE09 inhibited pathogen development (mycelial growth, sporulation, and zoospore germination) in I-plate (a plastic plate containing a center partition). In addition, germinated seeds treated with the compound (1-100 µg ml⻹) or the strain (10²-10¹° cells ml⻹) significantly reduced radicle infection by P. capsici without radicle growth inhibition. CONCLUSIONS: These results indicate that colonization of pepper root and rhizosphere by the Fl. johnsoniae strain GSE09, which can form biofilms and produce indolic compounds, biosurfactant, and 2,4-di-tert-butylphenol, might provide effective biocontrol activity against P. capsici. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first study demonstrating that the Fl. johnsoniae strain GSE09, as a potential biocontrol agent, can effectively protect pepper plants against P. capsici infection by colonizing the roots.
Subject(s)
Biological Control Agents , Capsicum/microbiology , Flavobacterium/physiology , Phytophthora/growth & development , Biofilms , Flavobacterium/genetics , Phenols/pharmacology , Phytophthora/drug effects , Plant Diseases/microbiology , Plant Roots/microbiology , RNA, Ribosomal, 16S/genetics , Rhizosphere , Soil Microbiology , Surface-Active AgentsABSTRACT
AIM: To investigate the configuration of C-shaped canals in mandibular second molars, canal wall thickness and the orientation of the thinnest area at 1-mm intervals from the canal orifice to the apex by using cone-beam computed tomographic (CBCT) images. METHODOLOGY: Three-dimensional CBCT images of 92 Korean mandibular second molars having C-shaped root canals were analysed to determine their configuration using a modification of Melton's classification, as well as the thinnest walls and their location. Associations between configuration type and distance from the canal orifice to the apex, as well as associations between the directional orientation of the thinnest root wall and distance from the canal orifice to the apex, were assessed by Fisher's exact test. Because serial measurements of minimum wall thicknesses were correlated with individual teeth, a mixed-effects analysis was applied. RESULTS: The most common configuration types were Melton's type I in the coronal region and Melton's type III in the apical region. Mean thicknesses of the thinnest root canal walls were 1.39 ± 0.38, 0.85 ± 0.25 and 0.77 ± 0.20 mm in the coronal, middle and apical regions, respectively. The thicker the root canal walls at the orifice region, the greater the decrease in thickness towards the apical region (P < 0.05), with the linguo-central root area being the thinnest. The pattern of decreasing thickness from the orifice to the apex formed a nonlinear cubic curve. CONCLUSIONS: The most prevalent configuration types were Melton's type I (coronal region) and type III (apical region). The linguo-central root area was the thinnest in C-shaped root canals of Korean mandibular second molars. These anatomical variations should be considered during surgical or nonsurgical endodontic procedures.
Subject(s)
Anatomic Variation , Cone-Beam Computed Tomography/statistics & numerical data , Dental Pulp Cavity/diagnostic imaging , Molar/diagnostic imaging , Adult , Anatomy, Cross-Sectional/statistics & numerical data , Biometry , Dental Pulp Cavity/anatomy & histology , Humans , Imaging, Three-Dimensional/methods , Mandible/diagnostic imaging , Middle Aged , Molar/anatomy & histology , Odontometry/statistics & numerical data , Republic of Korea , Retrospective Studies , Tooth Apex/anatomy & histology , Tooth Apex/diagnostic imaging , Tooth Crown/anatomy & histology , Tooth Crown/diagnostic imaging , Tooth Root/anatomy & histology , Tooth Root/diagnostic imaging , Young AdultABSTRACT
PURPOSE: During mastication, mechanical pressure from the dentition is transmitted to the trabecular bone of the mandible. The occlusal forces, which could thus affect condylar growth, vary with tooth loss, age, and sex. The trabecular bone of the mandibular condyle is denser in dentate subjects than in edentate subjects. However, since the different tooth groups (incisor, premolar, and molar) have different functions, they could exert different effects on the mandibular condyle. The aim of this study was to elucidate the bone quantity of the Korean mandibular condyle according to the presence of teeth using micro-computed tomography (micro-CT), thereby clarifying the influences of tooth presence on the condylar microstructure. METHODS: Thirty-one sides were scanned and reconstructed into a 3D structure using a micro-CT system. The specimen was sectioned vertically, passing through the medial and lateral poles of the mandibular condyle (P0) to enable measurement of the trabecular bone ratio. Likewise, three additional images, parallel with P0, were acquired. Mean and standard deviation values were calculated, and the t test, one-way ANOVA and post hoc analysis were performed to examine the differences among each group, classified according to the presence of teeth and according to sex. RESULTS: The density of the trabecular bone of the mandibular condyle was significantly associated with the presence of the molars, but not the incisors or premolars. There were significant differences between sexes. CONCLUSIONS: The present study has provided data regarding the bone quantity of the trabeculae of the mandibular condyle according to the presence or absence of teeth.
Subject(s)
Bone Density , Mandibular Condyle/diagnostic imaging , Tooth Loss/diagnostic imaging , Tooth/diagnostic imaging , X-Ray Microtomography/methods , Aged , Aged, 80 and over , Analysis of Variance , Bicuspid/diagnostic imaging , Cadaver , Dentition , Female , Humans , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Incisor/diagnostic imaging , Korea , Male , Mandible/diagnostic imaging , Middle Aged , Molar/diagnostic imaging , Sex DistributionABSTRACT
AIM: This article presents a case report of autogenous tooth transplantation to a site which had insufficient bone volume using a sinus lift and allogenic bone graft. SUMMARY: An ectopic, fully impacted premolar tooth was autotransplanted from its ectopic impaction site into its original site, where there was insufficient recipient vertical bone volume because of sinus expansion. The deciduous tooth was extracted from the recipient site, and the sinus membrane detached and elevated via the alveolar socket. Allogenic bone grafting was performed, and the impacted premolar was transplanted into the prepared site. To improve adaptation, the recipient site was prepared using a rapid prototype tooth model, a replica tooth which allowed complete socket preparation in advanced of the actual removal of the donor tooth. The donor tooth was fixed with sutures and maintained for 17 days to allow physiologic movement. Root canal treatment was initiated 24 days after autotransplantation, and an intra-canal medicament was used for 4 months. Canal filling was completed 5 months after autotransplantation. There was no root resorption of the transplanted tooth, and the grafted bone was well preserved and had no signs of infection. KEY LEARNING POINTS: When the recipient bone volume is insufficient, autotransplantation can be preceded by bony augmentation. The preparation of the recipient tooth socket using a tooth replica from CBCT reduces the extra-oral time of the actual tooth and promotes better periodontal ligament healing. Careful evaluation of the pulp status of the donor tooth is important in advance of timely endodontic treatment.
Subject(s)
Bicuspid/transplantation , Bone Transplantation/methods , Maxilla/surgery , Sinus Floor Augmentation/methods , Tooth Eruption, Ectopic/surgery , Tooth, Impacted/surgery , Adolescent , Calcium Hydroxide/therapeutic use , Dental Pulp Necrosis/etiology , Dental Pulp Necrosis/therapy , Follow-Up Studies , Graft Survival , Humans , Male , Postoperative Complications , Root Canal Irrigants/therapeutic use , Root Canal Obturation , Root Canal Therapy , Tooth Socket/surgery , Transplantation, Autologous , Transplantation, HomologousABSTRACT
Recent genomic studies have revealed that chromosomal structures are formed by a hierarchy of organizing processes ranging from gene associations, including interactions among enhancers and promoters, to topologically associating domain formations. Gene associations identified by these studies can be characterized by microscopic analyses. Fission yeast is a model organism, in which gene associations have been broadly mapped across the genome, although many of those associations have not been further examined by cell biological approaches. To address the technically challenging process of the visualization of associating gene loci in the fission yeast nuclei, we provide, in detail, an IF-FISH procedure that allows for covisualizing both gene loci and nuclear structural markers such as the nuclear membrane and nucleolus.
Subject(s)
Chromosomes, Fungal , Fluorescent Antibody Technique/methods , Genetic Loci , In Situ Hybridization, Fluorescence/methods , Schizosaccharomyces/genetics , Schizosaccharomyces/ultrastructure , Centromere/genetics , Centromere/ultrastructure , Chromosomes, Fungal/genetics , Chromosomes, Fungal/ultrastructure , Genome, Fungal , Schizosaccharomyces/cytologyABSTRACT
We showed previously that hypertriglyceridaemia, but not hypercholesterolaemia, is correlated with increases in cholesterol synthesis and apolipoprotein B secretion in patients with secondary hypertriglyceridaemia. The aim of the present study was to compare the rate of cholesterol synthesis, using fasting plasma mevalonic acid (MVA) as an index, in patients with primary mixed hyperlipidaemia (type IIb phenotype, n=45) and primary hypercholesterolaemia (type IIa phenotype, n=92). LDL cholesterol was significantly higher in types IIa (6.38+/-0.18 mmol/l) and IIb (5.89+/-0.25 mmol/l) compared to 40 normolipidaemic controls (2. 99+/-0.1 mmol/l, P<0.0001), whereas serum triglyceride was higher in type IIb (2.62 (range 2.2-3.0) mmol/l) than type IIa (1.22 (range 0. 85-1.60) mmol/l, P<0.001) and controls (0.90 (range 0.68-1.24) mmol/l, P<0.001). Similarly, MVA was higher in type IIb (7.0+/-0.46 ng/ml) than IIa (5.6+/-0.23 ng/ml, P<0.0) and controls (5.6+/-0.36 ng/ml, P<0.05). Plasma MVA correlated positively with serum triglyceride (r=0.22, P=0.004) and negatively with LDL cholesterol (r=-0.21, P=0.014). These results are in accordance with previous observations that VLDL-apolipoprotein B secretion and cholesterol synthesis are linked and demonstrate that the latter is increased in mixed hyperlipidaemia.
Subject(s)
Cholesterol/biosynthesis , Hyperlipidemias/blood , Adult , Apolipoproteins E , Cholesterol, LDL/blood , Fasting , Female , Humans , Hydroxymethylglutaryl CoA Reductases/metabolism , Hyperlipoproteinemia Type II/blood , Male , Mevalonic Acid/blood , Phenotype , Triglycerides/bloodABSTRACT
Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti-inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)-12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose- and time-dependent manner as it was measured by determining annexin V-positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex-mediated apoptosis. The Dex-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex-induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.
Subject(s)
Apoptosis/physiology , CD40 Antigens/physiology , Caspases/metabolism , Dendritic Cells/cytology , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Anti-Inflammatory Agents/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , Antibodies/immunology , Antibodies/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , CD40 Antigens/immunology , Caspase Inhibitors , Dendritic Cells/drug effects , Dendritic Cells/physiology , Dexamethasone/antagonists & inhibitors , Enzyme Activation , Gene Expression , Glucocorticoids/antagonists & inhibitors , Hormone Antagonists/pharmacology , Humans , Mifepristone/pharmacology , Receptors, Glucocorticoid/antagonists & inhibitors , Signal Transduction/physiologyABSTRACT
Distances from the apex to the buccal bone plate were measured on the computed tomography (CT) images of 1806 teeth from 66 patients, using an image analyzer program (Image-Pro Plus, Ver. 4.0, Media Cybernetics). In the mandible, the mean distance from the distal apex of the mandibular second molar to the buccal bone plate was the largest distance measured, at 8.51 mm, followed by distance from the mesial root to the buccal bone (7.34 mm). In the mandibular first molar, the mean distal and mesial bone thicknesses were 5.18 mm and 4.09 mm, respectively. However, when there were two distal roots, the distance of the disto-lingual root to the buccal plate was found to be 9.52 mm, which constitutes the greatest measured thickness. In the maxillary buccal roots, the distances from the mesio-buccal and disto-buccal root of the second molar to the buccal bone plate were the largest, at 4.63 mm and 3.61 mm, respectively. The average distances from the palatal apex of the maxillary first and second molars to the buccal bone plate were 10.69 mm and 10.17 mm, respectively, while, from the palatal bone plate, average distances of 3.15 mm and 3.08 mm were measured. Special considerations, such as bony lid approach, lingual approach, or intentional replantation may be required, especially when a patient has a surgical need in the second molars and the disto-lingual root of the mandibular first molar, or in the palatal root of the maxillary molars.
Subject(s)
Mandible/anatomy & histology , Maxilla/anatomy & histology , Adolescent , Adult , Aged , Aged, 80 and over , Asian People , Bone Density , Cheek , Female , Humans , Male , Mandible/diagnostic imaging , Maxilla/diagnostic imaging , Middle Aged , Reference Values , Tomography, X-Ray Computed , Tooth Apex/anatomy & histologyABSTRACT
The influence of hypothyroidism in the adult rat on brain biochemistry was investigated. Hypothyroidism was induced in 6-month-old male rats by partial thyroidectomy coupled with the administration of 6-n-propyl-2-thiouracil (0.005%, w/v) in the drinking water. Age-matched euthyroid males served as the controls. Hypothyroidism resulted in brain region-specific changes in certain catabolic enzyme activities. Acid phosphatase activity was reduced in the cerebellum (by 34%) and the medulla (by 38%), whereas alkaline phosphatase activity was decreased in the midbrain (by 37%) and the subcortex (by 49%). A differential response was also observed in the case of aryl sulphatase activity: aryl sulphatase A (myelin-degradative activity) was diminished in the cerebellum (by 56%), whereas aryl sulphatase B remained unchanged in all regions. Acetylcholine esterase activity was reduced in the cerebellum (by 45%), the medulla (by 34%) and the subcortex (by 45%), whereas monoamine oxidase activity was affected in only one region, the cerebellum, where it was increased by (61%). The compromise of myelin and neurotransmitter degradative enzyme activities may place severe restrictions on normal brain function. The vulnerability of the adult rat cerebellum to the effects of thyroidectomy is commensurate with the known clinical signs of cerebellar dysfunction in adult hypothyroid man. These findings raise the possibility of an important role for the thyroid hormones in the mature brain.
Subject(s)
Brain/enzymology , Hypothyroidism/enzymology , Acetylcholinesterase/metabolism , Acid Phosphatase/metabolism , Alkaline Phosphatase/metabolism , Animals , Cerebellum/enzymology , Cerebroside-Sulfatase/metabolism , Chondro-4-Sulfatase/metabolism , Disease Models, Animal , Male , Medulla Oblongata/enzymology , Rats , Rats, Sprague-Dawley , ThyroidectomyABSTRACT
A newly synthesized affinity ligand, (R,S)-5-isothiocyanonicotine (ISCN-N) was found to inhibit irreversibly the binding of [3H]methylcarbamylcholine (a specific nicotinic receptor ligand) to brain membranes. Plots of percent inhibition versus ligand concentration yielded an IC50 of 7 x 10(-8) M for SCN-N and Ki values of 6 x 10(-9) and 2 x 10(-9) M for (R,S)-5-aminonicotine and (S)-nicotine, respectively. The IC50 value for irreversible inhibition of [3H]methylcarbamylcholine by SCN-N was 2 x 10(-7) M. The affinity ligand irreversibly inhibited brain nicotinic receptors in vivo in a dose-dependent manner, the inhibition being 49% at a dose of 20 mumol/kg. Behavioral studies in mice revealed that SCN-N had less than one-fifth the potency of nicotine in producing muscle weakness and seizures, whereas 5-aminonicotine was without significant behavioral effects at doses up to 20 mumol/kg.
Subject(s)
Brain/drug effects , Nicotine/analogs & derivatives , Nicotinic Antagonists , Animals , Behavior, Animal/drug effects , Brain/metabolism , Carbachol/analogs & derivatives , Carbachol/metabolism , Cattle , Locomotion/drug effects , Mice , Nicotine/antagonists & inhibitors , Nicotine/chemical synthesis , Nicotine/pharmacology , Nicotine/toxicityABSTRACT
An Itm2C cDNA encoding a member of the mouse type II integral transmembrane protein (Itm2) was cloned from a mouse 13.5 days' post-coitum (dpc) embryonic head cDNA library. The nucleotide sequence and protein prediction analyses indicate that the membrane topology of the mouse Itm2c is a type II integral transmembrane protein with the N-terminus being intracellular and C-terminus, which is considered as either an extracellular or a luminal organelle domain. The predicted protein consists of 269 amino acids with a calculated Mr of 30,482 and a theoretical pI of 8.83. A putative N-glycosylation site was identified at amino acid 171, Asn. The single N-glycosylation site is conserved in the three members of the family in both mice and humans. The amino acid sequence of Itm2C is 41 and 49% identical to those of mouse Itm2A and Itm2B, respectively. A single approximately 2.1 kb transcript was detected with different levels in all of the tissues examined by Northern blot analysis. The Itm2c was highly expressed in both adult and postimplantation embryonic brains, although weak or moderate signals were also detected in other adult tissues. Among the tissues examined, RT-PCR analyses in various adult tissue shows that the Itm2c expression is highest in the adult brain. The results from this study suggest that the cloned Itm2c is a member of the Itm2 family that is specifically expressed in adult brains, unlike other members.
Subject(s)
Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Brain/metabolism , Cloning, Molecular , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Multigene Family , Nerve Tissue Proteins/metabolism , Organ Specificity , Sequence Alignment , Sequence Analysis, DNA , Sequence HomologyABSTRACT
The induction of pluripotent P19 embryonal carcinoma (EC) cells with retinoic acid results in their differentiation into cells that resemble neurons, glia, and fibroblasts. To isolate and enrich the developing neurons from heterogeneously differentiating P19 EC cells, we used a recently introduced protocol combining the expression of green fluorescent protein (GFP) driven by a tissue-specific promoter and fluorescence-activated cell sorting. Cells were transfected with the gene for GFP, which is under the control of the neuronal T alpha 1 tubulin promoter. After four days of retinoic acid treatment, GFP was specifically detected in cells undergoing neuronal differentiation. Sorting of fluorescent differentiating P19 EC transfectants yielded populations highly enriched in neuronal precursors and neurons. Immunoreactivity for nestin and neurofilament was observed in 80 and 25% of the sorted cell population, respectively. These results demonstrate that differentiated neuronal precursor cells can be efficiently isolated from differentiating pluripotent embryonic cells in vitro, suggesting that this method can reproducibly provide homogeneous materials for further studies on neurogenesis.
Subject(s)
Cell Differentiation/physiology , Luminescent Proteins/genetics , Neoplastic Stem Cells/cytology , Neurons/cytology , Promoter Regions, Genetic/physiology , Stem Cells/cytology , Tubulin/genetics , Antineoplastic Agents/pharmacology , Cell Culture Techniques , Cell Differentiation/drug effects , Clone Cells/cytology , Clone Cells/drug effects , Clone Cells/metabolism , Embryonal Carcinoma Stem Cells , Embryonic Induction/drug effects , Embryonic Induction/physiology , Flow Cytometry , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Green Fluorescent Proteins , Humans , Immunohistochemistry , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neurons/drug effects , Neurons/metabolism , Stem Cells/drug effects , Stem Cells/metabolism , Transgenes/drug effects , Transgenes/physiology , Tretinoin/pharmacologyABSTRACT
OBJECTIVE: To examine the patterns of submicroscopic DNA deletions in the AZF (AZoospermia Factor) subregions of the Y chromosome in patients with idiopathic azoospermia. DESIGN: Controlled clinical study. SETTING: University-based infertility clinic. PATIENT(S): Infertile men (n = 40) with nonobstructive, idiopathic azoospermia. The control group consisted of proven fathers (n = 14) and healthy women (n = 4). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen analysis; polymerase chain reaction amplification of the 37 loci spanning the AZFa, AZFb, and AZFc subregions of the Y chromosome; serum FSH, LH, and testosterone levels; and testicular histologic analysis. RESULT(S): Testicular histologic analysis of the subjects revealed Sertoli cell-only syndrome (n = 36) and spermatogenic arrest (n = 4). Microdeletions of the Y chromosome were found in eight (20%) of the patients with azoospermia. All eight affected patients had interstitial microdeletions within the AZFc subregion. Patients with Sertoli cell-only syndrome had additional microdeletions in regions distal to DAZ (Deleted in Azoospermia), although DAZ deletion was observed in seven of the eight affected patients. In five patients, microdeletions were found in the AZFb region containing RBM (RNA Binding Motif). CONCLUSION(S): Our results add to the evidence supporting the current suggestion that there is a cause-and-effect relation between Yq11 microdeletions in the AZF region and azoospermia.
Subject(s)
Oligospermia/genetics , Sequence Deletion , Y Chromosome , Adult , Chromosome Mapping , Female , Follicle Stimulating Hormone/blood , Humans , Luteinizing Hormone/blood , Male , Oligospermia/blood , Oligospermia/pathology , Polymerase Chain Reaction , Reference Values , Sertoli Cells/pathology , Spermatogenesis/genetics , Testis/pathologyABSTRACT
Immunization of dendritic cells (DC) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL) that are responsible for protection and regression. We show here that immunization with bone marrow-derived DC cocultured with tumor cells can induce a protective immunity against challenges to viable tumor cells. In this study, we further investigated the mechanism by which the antitumor activity was induced. Immunization of mice with DC cocultured with murine colon carcinoma. CT-26 cells, augmented CTL activity against the tumor cells. Concomitantly, an increase in natural killer (NK) cell activity was also detected in the same mice. When DC were fixed with paraformaldehyde prior to coculturing with tumor cells, most of the CTL and NK cell activity diminished, indicating that DC are involved in the process of presenting the tumor antigen(s) to CTL. NK cell depletion in vivo produced markedly low tumor-specific CTL activity responsible for tumor prevention. In addition, RT-PCR analysis confirmed the high expression of INF-gamma mRNA in splenocytes after vaccination with DC cocultured with tumors, but low expression in splenocytes from NK-depleted mice. Most importantly, the tumor protective effect rendered to DC by the coculturing with CT-26 cells was not observed in NK-depleted mice, which suggests that DC can induce an antitumor immune response by enhancing NK cell-dependent CTL activation. Collectively, our results indicate that NK cells are required during the priming of cytotoxic T-cell response by DC-based tumor vaccine and seem to delineate a mechanism by which DC vaccine can provide the desired immunity.
Subject(s)
Cancer Vaccines/immunology , Dendritic Cells/immunology , Killer Cells, Natural/immunology , T-Lymphocytes, Cytotoxic/immunology , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma/prevention & control , Adenocarcinoma/therapy , Animals , Antigen Presentation , Bone Marrow Cells/cytology , Coculture Techniques , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Colonic Neoplasms/therapy , Cytotoxicity, Immunologic , Dendritic Cells/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Immunization , Immunologic Memory , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-4/pharmacology , Kidney Neoplasms/pathology , Lymphocyte Activation , Lymphocyte Depletion , Lymphoma/pathology , Male , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Specific Pathogen-Free Organisms , Tumor Cells, Cultured/immunology , VaccinationABSTRACT
PURPOSE: To describe new software applications and interchangeable instrumentation enabling the use of standard surgical instruments with image-guided systems for stereotactic spinal procedures. CONCEPT: The ability to adapt essentially any surgical instrument for stereotactic procedures will improve the safety and accuracy of image-guided spinal surgery. RATIONALE: Using universal dynamic registration hardware and software, standard surgical instruments are adapted for real-time image-guided surgery. The Radionics Optical Tracking System (Radionics, Inc., Burlington, MA) has custom software applications and universal hardware adaptation devices for spinal stereotaxy that allows the use of standard instruments for intraoperative guidance. An array of light-emitting diodes can be attached to essentially any rigid instrument with a definable tip and can then be calibrated to the system for intraoperative use. Stereotactic guidance of a drill, tap, and screwdriver may improve screw placement accuracy in spinal surgery because every step of the procedure can be monitored in real time. DISCUSSION: Most stereotactic systems have only a standard probe or limited instruments for localization, targeting, and tracking a procedure. The surgeon then resumes the operation using standard surgical instruments without the benefit of image guidance for the key steps of the procedure. Because each surgical step for screw placement in the spine has a potential for error, use of multiple instruments that can be interchanged for real-time image-guided spinal surgery may increase the accuracy and safety of spinal instrumentation procedures. These techniques can also be applied to intracranial image-guided surgery.
Subject(s)
Image Processing, Computer-Assisted/instrumentation , Spinal Diseases/surgery , Stereotaxic Techniques/instrumentation , Surgical Instruments , Bone Screws , Calibration , Equipment Design , Humans , Spinal Fusion/instrumentationABSTRACT
STUDY DESIGN: In this cadaveric study, a computer-assisted image guidance system was tested for accuracy of thoracic pedicle screw placement. OBJECTIVES: Evaluate the system's accuracy for thoracic pedicle screw placement in vitro. SUMMARY OF BACKGROUND DATA: The effective use and reliability of pedicle screw instrumentation in providing short-segment stabilization and correction of deformity is well known in the lumbar spine. Pedicle screw placement in the thoracic spine is difficult because of the small dimensions of the thoracic pedicles and risk to the adjacent spinal cord and neurovascular structures. Investigators have shown the improved accuracy of computer-assisted lumbar pedicle screw placement; but the accuracy of computer-assisted thoracic pedicle screw placement, which is becoming more widely used, has not been shown. METHODS: In five human cadavers, 120 thoracic pedicle screws were placed with computer-assisted image guidance. The largest clinically feasible screw was used based on the cross-sectional dimensions of each pedicle. The accuracy was assessed by postoperative computed tomography and visual inspection. RESULTS: The overall pedicle cortex violation was 23 of 120 pedicles (19.2%). Nine violations (7.5%) were graded as major and 14 (11.7%) as minor. A marked and progressive learning curve was evident with the perforation rates that decreased from 37.5% in the first cadaver to 4.2% in the last two cadavers. CONCLUSIONS: Accurate thoracic pedicle screw placement is feasible with computer-assisted surgery. However, as with any other new surgical technology, the learning curve must be recognized and incorporated into the necessary fundamental knowledge and experience for these procedures.
Subject(s)
Bone Screws , Image Processing, Computer-Assisted/instrumentation , Image Processing, Computer-Assisted/methods , Spinal Diseases/surgery , Thoracic Vertebrae/surgery , Feasibility Studies , Humans , Intraoperative Complications/prevention & control , Spinal FusionABSTRACT
Spinal pseudomeningoceles and cerebrospinal fluid (CSF) fistulas are uncommon extradural collections of CSF that may result from inadvertent tears in the dural-arachnoid layer, traumatic injury, or may be congenital in origin. Most pseudomeningoceles are iatrogenic and occur in the posterior lumbar region following surgery. The true incidence of iatrogenic pseudomeningoceles following laminectomy or discectomy is unknown; however, the authors of several published reports suggest that the incidence of lumbar pseudomeningoceles following laminectomy or discectomy is between 0.07% and 2%. Pseudomeningoceles are often asymptomatic, but patients may present with recurrence of low-back pain, radiculopathy, subcutaneous swelling, or with symptoms of intracranial hypotension. Very rarely, they present with delayed myelopathy. Although magnetic resonance imaging is the neurodiagnostic study of choice, computerized tomography myelography and radionuclide myelographic study may be helpful diagnostic tools in some cases. Analysis of suspect fluid for Beta2 transferrin may be a useful adjunctive study. Treatment options include close observation for spontaneous resolution, conservative measures such as bed rest and application of an epidural blood patch, lumbar subarachnoid drainage, and definitive surgical repair.