Animals ; Bone and Bones ; cytology ; Cell Differentiation ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Neurons ; physiology ; Plant Extracts ; pharmacology ; Rats ; Salvia miltiorrhiza ; chemistry

OBJECTIVENeonatal hypoxic-ischemic encephalopathy (HIE) harms the lives and health of newborn infants and children severely. The prognosis is not satisfied, especially of the severe HIE. Mesenchymal stem cells (MSCs) can secrete a series of growth factors and neurotrophic factors. As well they have the potential ability to differentiate to the neural cells in vitro and in vivo. Therefore MSCs transplantation has been employed as a source of progenitor cells for cell therapy in patients with HIE in order to promote recovery of brain function and reduce the sequelae. Studies have shown that MSCs could enter the cerebral parenchyma and differentiate to neural cells through systemic infusion, but most of the researches applied adult stroke animal models. This study used neonatal HIE models to test the hypothesis that MSCs could enter the brain of newborn Wistar rats through the blood-brain barrier (BBB) by intraperitoneal infusion followed by observing the characteristics of the distribution and differentiation of MSCs in brain tissues, and exploring the effects of hypoxic-ischemic brain damage to the penetration and differentiation of MSCs.

METHODSIsolation and purification of MSCs were established from the whole bone marrow of juvenile Wistar rats by removing the nonadherent cells in primary and passage cultures. For cellular identification, MSCs of three to five passages were continuously pre-labeled with 5-bromo-2-deoxyuridine (BrdU) for 72 hours before transplantation. Animal models of HIE were built in 7-day-postnatal Wistar rats according to the method described by Rice. Two hours after hypoxia-ischemia, rats in HIE group (n = 8) were intraperitoneally infused with MSCs (4 x 10(6), 0.5 ml). In control group (n = 8), 7-day-postnatal normal Wistar rats were intraperitoneally infused with the same amount of MSCs. All rats were sacrificed and their cerebra were sectioned by cryomicrotome 14 days after transplantation. Immunohistochemical staining with chromogen diaminobenzidine (DAB) was used to detect and measure the cells derived from MSCs, and study the characteristics of distribution. To determine the differentiation of the BrdU positive cells entering the brains, immunofluorescence double labeling for BrdU and neural cells specific antigens was performed.

RESULTSMSCs were distributed throughout the cerebra in both groups at the 14th day after transplantation. The number of MSCs detected was 2415 +/- 226 in the control group, and 3626 +/- 461 in HIE group, respectively (t = 6.68, P < 0.05). More BrdU reactive cells were observed in the right ischemic hemisphere (1904 +/- 267) than in the contralateral hemisphere (1723 +/- 204), (t = 4.47, P < 0.05). No significant difference was found while comparing both cerebral hemispheres of the control group (t = 0.31, P > 0.05). In the HIE group, MSCs distributed more extensively, and some focal aggregations of MSCs were noticed. A few MSCs expressed Nestin-protein marker of neural progenitor cells, and almost none of the MSCs which expressed proteins characteristic of neuron (e.g. NSE) and astrocyte (e.g. GFAP) was detected at the 14th day after transplantation.

CONCLUSION1. MSCs could enter the cerebral parenchyma through BBB and migrate throughout the brain by intraperitoneal infusion. 2. More MSCs injected intraperitoneally were localized and directed to the sites of hypoxic-ischemic brain damage. 3. Transplanted MSCs could not differentiate to neuron and astrocyte without other interventions during 14 days after transplantation.

Animals ; Blood-Brain Barrier ; physiology ; Brain ; Cell Differentiation ; Cell Movement ; Disease Models, Animal ; Hypoxia-Ischemia, Brain ; therapy ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; physiology ; Rats ; Rats, Wistar

AIM: To examine the expression of CD69 on NK cells at the fetomaternal interface in CBA/J?DBA/2 mice as a model of recurrent spontaneous abortion (RSA), and to evaluate the effects of lymphocyte immunotherapy (LIT) on the level of CD69 expression and the relationship with the outcomes of murine fetuses and pups. METHODS: The outcomes of murine fetuses and pups were evaluated in breeding pairs of CBA/J?DBA/2, C57BL/6?DBA/2 and BALB/c?DBA/2 mice. Both preweaning growth curves and Kaplan-Meier survival graphs of pups were constructed throughout postnatal days 1 to 21. In addition, the level of CD69 expression on NK cells at the fetomaternal interface with and without LIT were determined by two-color flow cytometric analysis, stained with PE-CD69 and FITC-DX5. The subpopulation of CD16/CD32 + NK cells was also evaluated. RESULTS: Statistically significant differences were observed between CBA/J?DBA/2 mice and normal fertile controls in the median increase of maternal weight during pregnancy, the number of pups born per litter, the median neonatal weight on postnatal day 1, and the resorption rate of fetuses. The proportion of CD69 +DX5 + cells which represents activated NK cells was significantly higher in CBA/J?DBA/2 mice compared with normal fertile controls, while efficient LIT was able to dramatically decrease the expression of CD69 on NK cells at the fetomaternal interface and this was associated with the decrease of resorption rate accordingly. CONCLUSION: The fraction of CD69 +DX5 + cells seems to be functionally important in the mechanisms by which the embryos were rejected, whereas efficient LIT is capable of reducing the abortion rate via decreasing the expression of CD69 molecules on NK cells at the fetomaternal interface.

Amyloid Neuropathies, Familial ; genetics ; Cardiomyopathies ; Humans ; Mutation

BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.
B-Cell Activating Factor ; chemistry ; B-Cell Maturation Antigen ; biosynthesis ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Disulfides ; chemistry ; Escherichia coli ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Solubility ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; chemistry

OBJECTIVETo study the safety and effects of mini-margin nephron sparing surgery (NSS) for renal cell carcinoma (RCC).

METHODSFrom January 1998 to December 2006, 115 cases of RCC with diameter of 4 cm or less and stage of T1aN0M0 were treated with NSS using a margin of 5 mm or more. The mean diameter of the tumors was 3.3 cm (range 1.0-4.0 cm). Of the cases, 3 were with synchronous bilateral cancer while 112 cases were with normal opposite kidneys. The clinical results were followed and analyzed.

RESULTSAll of the operations were technically successful. The mean duration of surgical procedures was 90 min (ranged 80-120 min). The blood loss was 50 -200 ml. No patient needed blood transfusion. Renal arteries were occluded in 98 cases under hypothermic technique for a mean duration of 22 min (20-25 min). While in 17 cases, renal parenchyma squeezing was used for bleeding control. All of the 115 cases were of negative margin by weather frozen or routine pathologic study. The mean follow-up was 62 months (6-96 months). Local recurrence was found in 1 case during follow-up, with a local recurrence rate of 0.9%, while no distant metastasis was detected. All the patients were alive with no evidence of tumor bearing until last evaluation. Secondary gross hematuria occurred in 3 cases during hospital stay and cured by bed limitation. There were no major complications such as bleeding and urinary leakage or urinoma requiring re-operation.

CONCLUSIONSMini-margin nephron sparing surgery is likewise safe and effective in treating early localized renal cell carcinoma 4 cm or less. It provides excellent renal function preservation, favorable long-term progression-free survival, and is not associated with an increased risk of local recurrence.

Adult ; Aged ; Carcinoma, Renal Cell ; surgery ; Female ; Follow-Up Studies ; Humans ; Kidney Neoplasms ; surgery ; Male ; Middle Aged ; Nephrectomy ; methods ; Treatment Outcome

OBJECTIVETo investigate the diagnostic methods and reasonable treatment of Peutz-Jeghers syndrome (PJS).

METHODSClinical data of six patients with PJS were reviewed.

RESULTSRepeated abdominal pain, intussusception and intestinal polyp with bleeding were main manifestations. Four patients father,three patients grandfather and one patients mother were diagnosed with PJS. Three patients had family history of cancer. Case 4 and case 5 underwent laparotomy for many times because of intussusceptions caused by polyps or recurrent abdominal pain. Case 1 and case 4 had polyps synchronous with adenoma, and case 2 had polyp with gastric cancer. Main treatment included polyp resection and partial small intestinal and colon resection.

CONCLUSIONSPatients with PJS have family history of cancer and a high incidence of polyp recurrence of small intestine. Surgical intervention is the first choice regimen. Surveillance should be emphasized on gastrointestinal tract and other potential malignant organs in PJS patients.

Adolescent ; Adult ; Female ; Humans ; Intestine, Small ; surgery ; Male ; Pedigree ; Peutz-Jeghers Syndrome ; diagnosis ; genetics ; surgery

Objective To investigate the prognostic value of acute beart block (AHB) after percutaneous transluminal septal myocardial ablation (PTSMA) in patients with hypertrophic obstructive cardiomyopathy (HOCM). Methods Ninety-four HOCM patients underwent PTSMA were included in this study. Twelve-lead electrocardiograms were obtained during and post PTSMA. Association between clinic events and incidence of post-PTSMA AHB was analyzed. Results AHB was induced in 26 patients by PTSMA and disappeared in 11 patients shortly post PTSMA, subacute intraventricular conduction disturbances was seen in 11 (42.3%), subacute I°AVB in 2(7.7% ) and subacute Ⅲ°AVB in another 2 (7.7%) patients. Among 68 patients without AHB during PTSMA, intraventricular conduction disturbances was evidenced in 14 patients (20.6% ), I°AVB in 2 (2.9% ) and Ⅲ°AVB in 1 patient (1.5%) after PTSMA. AHB patients with subacute heart block were associated with poor prognosis (conduction block duration was 42.00 h) while patients without AHB was associated with benign prognosis even with new onset of subacute heart block (conduction block duration was 7.33 h, P <0.01). Conclusion Patients with AHB during PTSMA are at higher risk for subacute heart block, especially intraventricolar conduction disturbances. AHB patients with subacute heart block were associated with poor prognosis and longer recovery time conducting system.

OBJECTIVEMechanical ventilation support is a very important method for the salvage of serious patients. However, it can result in the formation of an adherent matrix of bacteria on the surfaces of implanted materials which is termed "biofilm". Biofilm is dense bacterial communities attached to a solid surface and surrounded by an exopolysaccharide matrix. One of the most important features of bacterial biofilm is their resistance to antimicrobial agents and host immune system components. As a consequence, diseases involving biofilm are generally chronic and difficult to treat. The present study was conducted to explore the relationship between ETT-biofilm and the lower respiratory infection by observing microbial colonization and associated biofilm accumulation on the surface of endotracheal tubes (ETTs) removed from neonates treated with intubated mechanical ventilation.

METHODSTwenty neonates undergoing mechanical ventilation (from January to June in 2005) were recruited into this study. Clinical data about lower respiratory infection for each case were collected. ETTs were collected at the first time of extubation. A sterile control tube was also processed. For each ETT, a 1-cm-long cross-sectional segment was divided into two portions for both scanning electron microscopy (SEM) and aerobic/anaerobic cultures. The presence of biofilm on the surface of ETTs were examined by SEM, meanwhile, bacteria harvested from the surface of ETTs and the secretions of lower respiratory tract were isolated, identified and assessed on antimicrobial susceptibility, respectively.

RESULTSThe diagnosis on admission of the twenty cases included: neonatal respiratory distress syndrome (10), meconium aspirate syndrome (2), severe asphyxia (2), pneumatothorax (2), severe pneumonia (1), scleredema neonatorum (1), inborn pulmonary hypoplasia (1) and recurrent apnea (1). Thirteen cases did not present symptoms and signs of lower respiratory infection before mechanical ventilation. However, during the mechanical ventilation process, symptoms and signs of lower respiratory infection presented and lasted until extubation. Nine of the above mentioned thirteen cases (70%) had the same duration of tube use as mechanical ventilation duration (mean: 3.6 days). Observation by SEM showed that colonization was time dependent and the incidence of microbial colonization increased when the duration of tube use exceeded one days (12/20). There were no obvious bacterial colonies except that some amorphous material was noted in 5 of 20 ETTs as early as one day of tube use. Up to 2 days of tube use (4/20), attached bacterial colonization was seen embedded in amorphous material (3/4). Up to 3 days (7/20), a layer of biofilm formation presented on ETTs (5/7). Furthermore, biofilm architecture became more mature and complex if the duration exceeded 3 days. Neither bacteria nor biofilm formation was seen on the control ETT. The results of aerobic/anaerobic cultures showed that there were 14 cultures from ETTs (normal flora grew in 4) and 7 pathogens were isolated; 13 cultures from the secretions of lower respiratory tract (normal flora grew in 1) and 10 pathogens were isolated. Seven samples had the same pathogen both on the surface of ETTs and in the secretions of lower respiratory tract, which accounted for 50% of the positive cultures from ETTs, including Xanthomonas maltophilia (2), Klebsiella pneumoniae (2), Acinetobacter lwoffii (1), Acinetobacter baumannii (1) and normal flora (1). The gram-negative bacteria isolated from the surface of ETTs and the secretions of lower respiratory tract presented multi-resistance to antibiotics.

CONCLUSIONSThe ETT-biofilm develops into mature and complex form with the duration of tube use increase. This study provides evidence that there is correlation between microbial colonization, biofilm formation on the surface of ETTs and the lower respiratory infection in neonates who were intubated and ventilated for a prolonged period. ETT-Biofilm could also be a possible source of the recurrent infection. Increased attention must be paid to modification of the ETT to prevent or substantially reduce biofilm formation.

Acinetobacter baumannii ; isolation & purification ; Anti-Bacterial Agents ; adverse effects ; therapeutic use ; Biofilms ; Colony Count, Microbial ; Equipment Contamination ; Female ; Gram-Negative Bacteria ; Humans ; Infant ; Infant, Newborn ; Intubation, Intratracheal ; adverse effects ; Male ; Microscopy, Electron, Scanning ; methods ; Pediatrics ; Pneumonia ; drug therapy ; etiology ; Respiration, Artificial ; adverse effects ; Respiratory Tract Infections ; drug therapy ; etiology ; Trachea ; microbiology

OBJECTIVETo investigate the optimal margin in nephron-sparing surgery (NSS) for renal cell carcinoma (RCC) 4 cm or less in diameter.

METHODSEighty-two kidneys with RCC 4 cm or less in diameter resected by radical nephrectomy were prospectively studied. The kidney samples were sectioned at 3 mm interval and examined for multicentricity. On each layer of tissue sectioned, parenchyma margin of 15 mm beyond pseudocapsule was continuously sectioned and examined for completeness of pseudocapsule and extra-pseudocapsule cancer lesion. The farthest distance between extra-pseudocapsule lesion and primary tumor was measured. PCNA expression was detected in 41 patients by using standard SP immunohistochemistry technique.

RESULTSThe diameter of 82 primary tumors was 3.4 +/- 0.8 cm (range 1.5 - 4.0 cm). Of these, 31.7% (26/82) were found without intact pseudocapsule and 17.1% (14/82) with positive cancer lesions beyond pseudocapsule. The average distance between extra-pseudocapsule cancer lesion and primary tumor was 0.5 +/- 1.3 mm (range 0 - 5.0 mm), with a confidential interval (CI) of 95% (0.11, 0.94). Statistically, the one side percentile P(95) was 4.9 mm, P(97.5) was 5.0 mm and P(100) was 5.0 mm. The mean PCNA index in the 41 patients with RCC was (29.5 +/- 17.6)%, which was (49.6 +/- 21.5)% in the group with extra-pseudocapsule cancer lesions and (24.6 +/- 12.7)% in the group without (t = 3.162, P = 0.013). The ratio of strong expression was 5/8 in the group with extra-pseudocapsule cancer lesions, and 18.2% (6/33) in the group without the lesions (chi(2) = 6.442, P = 0.011). Logistic regression analysis showed that completeness of pseudocapsule and PCNA index were significant predictors of extra-pseudocapsule cancer lesions (P = 0.019).

CONCLUSIONSThese data suggest that when NSS is performed in RCC 4 cm or less in diameter, a margin of more than 5 mm of adjacent parenchyma should be excised with the tumor. Enucleation alone was associated with a significant risk of incomplete excision, and therefore liable for local recurrence. Tumors with incomplete pseudocapsule and(or) high PCNA indices are more likely to have extra-pseudocapsule cancer lesions, so intensive follow-up is necessary after NSS.

Adult ; Carcinoma, Renal Cell ; metabolism ; pathology ; surgery ; Female ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; surgery ; Male ; Middle Aged ; Nephrectomy ; methods ; Proliferating Cell Nuclear Antigen ; metabolism ; Retrospective Studies