Objective To establish a method for culturing neonatal mice cardiomyocytes, and construct an in vitro model of cardiomyocytes for assessing the cardiac toxicity of chemicals. Methods Hearts of neonatal mice of 1 day old were digested with enzyme mixture of trypsin/collagenase type Ⅱ/dispase and the cell suspensions were pre-plated to flask for a short time and then seeded on coated dishes. The cultured cells were treated with 5-fiuorine-deoxy-uridine(15 μg/ml)and uridine(35 μg/ml)to enrich cardiomyocytes that were identified according to the morphology and immunocytochemistry of α-actin antigen. Cardiac myocytes were incubated with 0-64 μg/ml of a fusarium mycotoxin butenolide(BUT) for 12 h. Cell viability was then evaluated by MTT assay. Microscopic observation showed that BUT induced significant morphological changes including cellular swelling, vacuolation and breakage of muscle fibers. Results It was found that 96% of the cultured cells were cardiomyocytes and the myocytes kept beating after 90 days of culture. Concentration-dependent decreases in cell viability following exposure of cardiac myocytes to BUT were observed. Conclusion The results indicated that relatively pure primary culture of neonatal mice cardiomyocytes is successfully established. BUT possesses the potential to induce myocardial toxicity.

Objective To observe and evaluate acute toxicities in a series of human immunodeficiency virus (HIV)-positive cancer patients receiving radiation therapy.Methods The study retrospectively reviewed the acute radiation reaction of radiation therapy of 14 HIV seropositive patients diagnosed with carcinoma between Feb 2008 and Dec 2013 at the Yunnan Tumor Hospital during the radiotherapy period and 1 month following treatment.Acute adverse effects were classified according to the site of radiation therapy and analyzed using the Common Terminology Criteria for Adverse Events (CTCAE) version 3.0.Results Seven patients experienced interruptions or delays in treatment,and 2 stopped treatment entirely.The most common acute adverse effects were skin reactions and mucous membrane reactions,including dermatitis,stomatitis or diarrhea.Eight patients had grade 3 acute adverse effects,including 6 patients with grade 3 skin reactions and 2 patients with grade 3 mucosa reactions.Conclusions Radiotherapy is an effective treatment for HIV positive patients with tumors,however it frequently induced severe acute radiation responses.

OBJECTIVEWernicke's encephalopathy (WE) is an acute neuropsychiatric syndrome resulting from thiamine deficiency, which is associated with significant morbidity and mortality. The disorder is still greatly underdiagnosed in children because of either a relatively non-specific clinical presentation in some cases or unrecognized clinical setting. The aim of this literature review was to provide knowledge of pediatric WE in an effort to assist in early diagnosis, thereby reducing the morbidity and mortality.

METHODSThe clinical manifestations, characteristic magnetic resonance imaging (MRI), diagnosis and treatment of one case and the other 35 cases reported in the last decade in children were summarized.

RESULTSThirty-six cases (22 boys and 14 girls, 2-month to 16-year-old) were analyzed. All the other 35 cases except for our case had underlying diseases: improper feeding in 25/35 cases, long-time vomiting in 5/35 cases, immunosuppressive therapy in 4/35 cases, long-time total parenteral nutrition without multivitamin preparations supplementation in 3/35 cases and anorexia nervosa in 1/35 case. The classic triad (mental-status changes, nystagmus and ophthalmoplegia, and ataxia) was seen in 6/36 cases. The other clinical manifestations included consciousness disturbance in 24/36 cases, infection in 22/36 cases, pathological reflex and muscular tension changes in 18/36 cases, convulsion in 17/36 cases, developmental delay in 4/36 cases and failure to thrive in 2/36 cases. Cerebrospinal fluid examination was performed in 31/36 cases, and a slightly raised protein concentration was seen in 7/31 cases. The cerebrospinal fluid lactate levels were detected in 4/36 cases (all increased), serum lactic acid levels in 7/36 cases (6/7 cases increased), serum pyruvate in 4/36 cases (all increased), thiamine pyrophosphate effect (TPPE) in 9/36 cases (all increased), and serum thiamine in 2/36 cases (increased in 1/2 cases). The brain computed tomography (CT) scan was conducted in 20/36 cases and 16/20 cases showed abnormal hypodensity in bilateral basal ganglia, one case revealed diffuse cortical atrophy. The brain MR scan was conducted in 13/36 cases and all the 13 cases revealed symmetrical abnormal signal in bilateral mamillary body and basal ganglia, and 7/13 cases showed abnormal signals in the tegmentum of midbrain, cerebral aqueduct and white matter around the third and fourth ventricles. The diagnosis of WE was confirmed by MR in 12 cases, triad combined with MR in 3 cases, autopsy in 1 case among the 13 cases who underwent MR scan. The diagnosis of WE was confirmed by the TPPE and/or lactate levels in 9/11 cases. The initial thiamine was given by intravenous or intramuscular infusion in 33/36 cases, unknown method in 1 case, orally in 1 case and no thiamine was used in 1 case. The dosage of thiamine was 100 mg daily in 29/35 cases, unknown in 3/35 cases, 50 mg daily in 2/35 cases, 600 mg daily in 1/35 case. 34/35 patients' clinical symptoms improved during 24 hours to 1 week after initial treatment, and 1 case died due to no response to thiamine. Nineteen patients were followed up for 2-2.5 months and 17 cases recovered completely.

CONCLUSIONWernicke's encephalopathy can be difficult to diagnose because of a relatively non-specific clinical presentation. The characteristic MRI findings and the dramatic response of neurological signs to parenteral thiamine will assist early clinical diagnosis. Early and timely thiamine supplementation could reverse the clinical features and improve the prognosis in most cases.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Sepsis ; complications ; Wernicke Encephalopathy ; complications ; diagnosis

OBJECTIVETo explore the role of exogenous connective tissue growth factor (CTGF) in the collagen III synthesis of human renal tubular epithelial cell line HK2 in vitro.

METHODSCultured HK2 cells were randomly assigned to three groups: placebo-control, low-dose CTGF-treated (2.5 ng/mL) and high-dose CTGF-treated groups (20 ng/mL). Cell morphological changes were observed under an inverted microscope. Collagen III alpha mRNA expression was detected using RT-PCR. Immunohistochemistry staining was used to assess the levels of intracellular collagen III alpha protein.

RESULTSAfter 48 hrs of low- or high- dose CTGF treatment, the appearances of HK2 cells were changed from oval to fusiform. High-dose CTGF treatment increased collagen III alpha mRNA expression (0.4461+/-0.0274 vs 0.2999+/-0.0115; P<0.05) as well as the protein expression of collagen III alpha (0.4075+/-0.0071 vs 0.3503+/-0.0136; P<0.05) compared with the placebo-control group.

CONCLUSIONSCTGF can induce morphological changes of human renal tubular epithelial cells in vitro. High concentration of CTGF may increase the synthesis of collagen III alpha.

Cells, Cultured ; Collagen Type III ; biosynthesis ; genetics ; Connective Tissue Growth Factor ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Immediate-Early Proteins ; pharmacology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; RNA, Messenger ; analysis

BACKGROUNDOur previous study has confirmed that one bout of exhaustion (Ex) can cause hippocampus neurocyte damage, excessive apoptosis, and dysfunction. Its initial reason is intracellular calcium overload in hippocampus triggered by N-methyl-D-aspartic acid receptor (NMDAR) over-activation. NMDAR activation can be suppressed by γ-aminobutyric acid (A) receptor (GABAAR). Whether GABAAR can prevent intense exercise-induced hippocampus apoptosis, damage, or dysfunction will be studied in this study.

METHODSAccording to dose test, rats were randomly divided into control (Con), Ex, muscimol (MUS, 0.1 mg/kg) and bicuculline (BIC, 0.5 mg/kg) groups, then all rats underwent once swimming Ex except ones in Con group only underwent training. Intracellular free calcium concentration ([Ca2+]i) was measured by Fura-2-acetoxymethyl ester; glial librillary acidic protein (GFAP) and synaptophysin (SYP) immunofluorescence were also performed; apoptosis were displayed by dUTP nick end labeling (TUNEL) stain; endoplasmic reticulum stress-induced apoptosis pathway was detected by Western blotting analysis; Morris water maze was used to detect learning ability and spatial memory.

RESULTSThe appropriate dose was 0.1 mg/kg for MUS and 0.5 mg/kg for BIC. Ex group showed significantly increased [Ca2+]i and astrogliosis; TUNEL positive cells and levels of GFAP, B cell lymphoma-2 (Bcl-2) associated X protein (Bax), caspase-3, caspase-12 cleavage, CCAAT/enhancer binding protein homologous protein (CHOP), and p-Jun amino-terminal kinase (p-JNK) in Ex group also raised significantly compared to Con group, while SYP, synapse plasticity, and Bcl-2 levels in Ex group were significantly lower than those in Con group. These indexes were back to normal in MUS group. BIC group had the highest levels of [Ca2+]i, astrogliosis, TUNEL positive cell, GFAP, Bax, caspase-3, caspase-12 cleavage, CHOP, and p-JNK, it also gained the lowest SYP, synapse plasticity, and Bcl-2 levels among all groups. Water maze test showed that Ex group had longer escape latency (EL) and less quadrant dwell time than Con group; all indexes between MUS and Con groups had no significant differences; BIC had the longest EL and least quadrant dwell time among all groups.

CONCLUSIONSActivation of GABAA R could prevent intense exercise-induced synapses damage, excessive apoptosis, and dysfunction of hippocampus.

Animals ; Apoptosis ; physiology ; Body Weight ; physiology ; Endoplasmic Reticulum Stress ; physiology ; Hippocampus ; metabolism ; Male ; Physical Exertion ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA ; genetics ; metabolism ; Synapses ; pathology

AIMTo investigate the effects of hypoxia on the proliferation of mouse embryonic stem cells (mouse ES cells) in vitro.

METHODSWe observed the proliferation of ES cells by hematometery and BrdU-labeled flow cytometry (FCM), and we also detected the expression of hypoxia inducible factor-1a (HIF-1a) by RT-PCR.

RESULTS(1) The number of ES cells after culturing in the hypoxia environment (3% O2 and 10% O2) for 24 hours were lesser than those in normoxia (20% O2). (2) The number of ES cells significantly increased after intermittent hypoxia (3% O2) stimulus for 10 minutes per day for 4 days. (3) We also observed the relation between the expression of HIF-1a and the proliferation of ES cells by RT-PCR. The results showed that the expression of HIF-1a had no significant change after ES cells were culturing in hypoxia environment (3% O2 and 10% O2) for 24 hours or in intermittent hypoxia (3% O2 and 10% O2) for 4 days.

CONCLUSIONThese results suggest that intermittent hypoxia (3% O2) can significantly promote the proliferation of ES cells in vitro, while persistent hypoxia inhibits those, and the mechanism of these should be addressed in further.

Animals ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Mice

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Glycyrrhizic Acid ; pharmacology ; Humans ; Lipopolysaccharides ; toxicity ; Liver ; drug effects ; pathology

OBJECTIVETo study and identify the protein markers in the urine of children with steroid-sensitive (SSNS) and steroid-resistant nephrotic syndrome(SRNS).

METHODSTotal urinary proteins were extracted from children with SSNS before and after steroid therapy, SRNS, and healthy children (n=5 in each group). Urinary proteins were separated by immobilized pH gradient based on two-dimensional gel electrophoresis (2-DE). The silver-stained 2-DE gels were scanned with digital Image Scanner and analyzed with Image Master 2-DE Elite 3.01 software. Peptide mass fingerprint (PMF) of differential protein spots was obtained with MALDI-TOF-MS. Proteins were identified by Mascot software based on NCBI protein database.

RESULTSThere were 66 spots with different expression of protein between SRNS children and SSNS children before steroid therapy, and 24 spots and 27 spots only occurred in SRNS children and SSNS children before steroid therapy, respectively. There were 75 spots with different expression of protein between SSNS children after steroid therapy and healthy controls, and 11 spots only occurred in SSNS children after steroid therapy. Eighteen protein spots with different expression (6 spots in each nephrotic group) were chose and analyzed by MALDI-TOF-MS, and 9 types of proteins were identified.

CONCLUSIONSNine types of urinary proteins with different expression (6 spots in each nephrotic group) were identified between SRNS and SSNS children, and they might be the biomarkers for SRNS or SSNS.

Adrenal Cortex Hormones ; therapeutic use ; Child ; Drug Resistance ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Nephrotic Syndrome ; drug therapy ; urine ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; alpha 1-Antitrypsin ; urine ; bcl-X Protein ; genetics

Angiotensin-Converting Enzyme Inhibitors ; therapeutic use ; Anticoagulants ; therapeutic use ; Cyclophosphamide ; therapeutic use ; Cyclosporine ; therapeutic use ; Fish Oils ; therapeutic use ; Glomerulonephritis, IGA ; therapy ; Glucocorticoids ; therapeutic use ; Humans ; Mycophenolic Acid ; analogs & derivatives ; therapeutic use

Objective To evaluate the role of cerebral state index(CSI),burst suppression (BS)and electromyograph(EMG)in monitoring coma/consciousness depth and damage degree of brain. Methods CSM was done in 50 cases with brain injury and coma to analyze its relation with physical reflection,auditory evoked potential(AEP),Glasgow coma score(GCS)and Glasgow outcome scale (GOS).Results As scale range meaning from consciousness to deep coma and to brain death,CSI 0- 100 was positively correlated with coma depth,coma score of GCS and physical reflection.CSI changes under invariable ache stimulation in combination with BS and EMG can accurately estimate prognosis and quantify changes of brain function.Conclusions The quantifiable digit of coma/consciousness depth and damage degree in brain function by CSM can attain real time judgment of dynamic evolvement course of coma and objective guide clinical therapy and assure prognosis,as will change absolutely scoring coma/ consciousness depth and prognosis under current state of artificial diversity and lacking objective evi- dences.