OBJECTIVE: To establish the method to analyze γ-hydroxybutyric acid (GHB) and its precursors 1,4-butanediol (1,4-BD) and gamma-butyrolactone (GBL) in urine through LC-MS/MS and provide evidence for related cases. METHODS: GHB-d6 and MOR-d3 were used as the internal standard. The urine sample was separated by LC after protein precipitation with methanol. The electrospray ion source was for ionization. Each compound was detected through multiple-reaction monitoring (MRM) mode. RESULTS: The limits of detection of GHB and its precursors 1,4-BD and GBL were 0.1, 0.1 and 2 μg/mL. The accuracy was 87.6%-98.1%. The intra-day and inter-day precisions were less than 15% and matrix effects were higher than 80%. CONCLUSION The method is high sensitive, simple, rapid, specific and with high reliability. This study has provided technical support and basic data for forensic cases involving GHB.
4-Butyrolactone/urine* ; Butylene Glycols/urine* ; Chromatography, Liquid ; Forensic Sciences ; Humans ; Hydroxybutyrates/urine* ; Mass Spectrometry ; Reproducibility of Results ; Tandem Mass Spectrometry

Objective To observe the position and mobility of the tape after tension-free vaginal tape-obturator(TVT-O) surgery using transperineal two and three dimensional ultrasonography in patients with stress urinary incontinence.Methods A total of 32 patients with stress urinary incontinence who had TVT-O surgery and cured were enrolled.Transperineal two and three dimensional ultrasonography were performed after operation 3 months later.Both the position,morphology and mobility of the tape at rest and during Valsalva menuaver were observed.Results The tapes were mainly located in the 1/3 connection of the middle and distal urethra.The length of the urethra and the length of the urethra between the bladder neck and the proximal tape at valsalva menuaver were longer than those at rest.The tapes moved front-down at valsalva manuaver according to the mean distance between the tape and the symphysis pubis.The angles of the tapes were smaller at valsalva manuaver without significant difference.Conclusion It is useful to observe the position,morphology and mobility of the tape using transperineal two and three dimensional ultrasonography.The changes of the position and morphology of the tapes at valsalva menuaver were not significant.But the tapes can strengthen the urine control in the middle of urethra.

OBJECTIVETo investigate the value of direct sequencing of sex-determining region Y (SRY) gene, as well as peripheral blood karyotype analysis, in the diagnosis of disorders of sex development (DSD) among children and adolescents with ambiguous genitalia.

METHODSThe karyotypes of 20 children and adolescents with ambiguous genitalia were determined by conventional G-banding analysis. PCR amplification was used to detect SRY gene in these patients, and direct sequencing was used to judge whether there was SRY gene mutation.

RESULTSOf the 20 cases, 17 were positive for SRY gene, and 3 were negative for SRY gene. Direct sequencing revealed no SRY gene mutation in the positive cases, however karyotype analysis found 4 special karyotypes in these patients: 46, XY, del(Y) (q12)/45, X; 46, XY, add(Y) (p11); 46, XY, r(9); 46, XY, 9qh+.

CONCLUSIONSSRY gene detection can help determine the type of DSD among children and has the advantage of quick detection. Used together with G-banding analysis, it is helpful for primary diagnosis of DSD among children.

Adolescent ; Child ; Child, Preschool ; Chromosome Banding ; Disorders of Sex Development ; diagnosis ; genetics ; Humans ; Infant ; Infant, Newborn ; Karyotype ; Sex-Determining Region Y Protein ; genetics

OBJECTIVETo investigate the clinical features, diagnostic and therapeutic strategy of pancreatic acinar cell carcinoma.

METHODSThe data of pancreatic acinar cell carcinoma patients who underwent surgical operations from January 2002 to January 2012 were retrospectively reviewed.

RESULTSSix cases of pancreatic acinar cell carcinoma, identified with pathology were collected, including 3 males and 3 females with the average of 47.8 yeas old. Upper abdominal pain was present in 5 cases, weight loss was present in 4 cases with the average of 12.5 kg. Other symptoms included nausea/vomiting, back pain and obstructive jaundice. The serum CA19-9 and CA24-2 level were significantly elevated in 2 cases. CT scan, MRI and DSA were the main imaging methods to diagnose this disease. However, no case was diagnosed as pancreatic acinar cell carcinoma before operation. All cases were confirmed by the pathological examination. Relatively high rates of surgical resection, long operative time, more blood loss and combined multi-organ resection were the characteristics of this disease's operative surgical procedures. The average period of postoperative follow-up process was 60 months, and the mean survival time was (32 ± 8) months.

CONCLUSIONSThe clinical features and biological behavior of pancreatic acinar cell carcinoma are different from those of ductal adenocarcinoma, while the relatively specific clinical manifestations and imaging changes will be helpful for qualitative diagnosis before operation. As it has high rate of resection and better prognosis, more radical surgical strategies should be carried out for patients of this disease.

Adult ; CA-19-9 Antigen ; blood ; Carcinoma, Acinar Cell ; diagnosis ; surgery ; Female ; Humans ; Male ; Middle Aged ; Pancreatic Neoplasms ; diagnosis ; surgery ; Prognosis ; Retrospective Studies ; Survival Rate

Objective To evaluate the effect of health education in controlling the iodine deficiency diserders(IDD) in order to provide reference data for the further prevention and control. Methods Each village of 3 towns in Congjiang County was selected in 2007, where the health education lasting for 10 months had been implemented in the school students of 3-6 grade and the villagers. The school students of 3-6 grade and 30 housewives in the villagers were investigated for their IDD control knowledge, the salt consuming conditions as well as the sales of both rough and fine salt at a salt retail site in each village before and after the health education was implemented. Results The awareness rate of the knowledge of IDD control in the students and housewives was 91.4% (581/636) and 78.3% (282/360), respectively after intervention, which significantly increased (χ2= 532.044, 326.117, both P < 0.01) compared with the rate of 28.2% (184/652) and 11.4% (41/360) before intervention. The proportion of consuming fine salt was 91.8%(146/159) and 95.6%(86/90), significantly inereased(χ2= 236.623, 135.350, both P < 0.01) compared with 6.1%(10/163) and 7.8% (7/90) found before intervention. The selling proportion of fine salt at the salt retail site in the village was 60.0%(900/1500), significantly increased(χ2= 824.176, P < 0.01) compared with 10.0%(150/1500) before intervention. Conclusions Health education and promotion is solid foundation for effectively controlling IDD, through which the students and villagers are actively and voluntarily involved in the program and hence have formed good living and hygiene habits, thus expected effect has been obtained.

Objective: To investigate the effect of brain derived neurotrophic factor (BDNF) on mesenchymal stem cells (MSC) inhibiting follicular helper T cells (Tfh cells). Methods: The contents of indoleamine 2,3-dioxygenase (IDO), IL-10, TGF-β and IL-21 in MSC culture supernatant were detected by ELISA; The peripheral blood of healthy volunteers were collected, and lymphocyte in peripheral blood was separated by human lymphocyte separation solution; Co-cultures of MSC and lymphocyte were performed by Transwell chamber, and the proportion of CD4(+)CXCR5(+) Tfh cells and their subtypes were detected by flow cytometry. Results: ①The concentrations of IL-10, TGF-β, and IDO in the supernatant of BDNF group (BDNF-stimulated MSC) were higher than those of the control ones (adding PBS with the same volume) [IL-10: (42.1±4.4) ng/ml vs (19.3±2.1) ng/ml, t=4.761, P=0.009; TGF-β: (13.9±1.7) ng/ml vs (5.3±0.6) ng/ml, t=5.129, P=0.008; IDO: (441.3±56.9) ng/ml vs (226.7±37.6) ng/ml, t=3.130, P=0.035]; ②The comparisons between BDNF (co-culture of lymphocyte and BDNF-stimulated MSC) and MSC groups (co-culture of lymphocyte and MSC) were detailed as of follows: the proportion of CD4(+) CXCR5(+)Tfh cells were lower [(3.37±0.21)% vs (6.51±0.27)%, t=9.353, P<0.001], the proportion of CD4(+) CXCR5(+)CXCR3(+) CCR6(-) Tfh cells were higher [(41.14±2.04)% vs (26.72±2.57)%, t=4.383, P=0.012], CD4(+)CXCR5(+)CXCR3(-)CCR6(-) Tfh2 cells and CD4(+)CXCR5(+)CXCR3(-)CCR6(+) Tfh17 cells were lower [Tfh2: (30.16±5.38)% vs (43.26±4.11)%, t=4.426, P=0.012; Tfh17: (15.61±1.52)% vs (22.32±0.72)%, t=4.202, P=0.014], the proportion of CD4(+)CXCR5(+)Foxp3(+) Tfr cells were higher [(4.95±0.22)% vs (2.32±0.16)%, t=10.241, P<0.001], the concentration of IL-21 in the lymphocyte supernatant was lower [(0.28±0.03) ng/ml vs (0.85±0.08) ng/ml, t=6.675, P=0.003]. Conclusion: BDNF could enhance the inhibitory effect of MSC on Tfh cells through inhibiting the increasing of Tfh cells and the differentiations of Tfh2 and Tfh17 cells.
Brain-Derived Neurotrophic Factor ; Cell Differentiation ; Flow Cytometry ; Humans ; Mesenchymal Stem Cells ; T-Lymphocytes, Helper-Inducer

HCAP1 is a novel hepatic cancer related gene located on human chromosome 17p13.3. The loss of heterozygosity occurred at 17p13.3 in various human cancers. In order to investigate the effects of exogenous HCAP1 gene products on cell proliferation of T lymphoma Jurkat cell line, HCAP1 gene! was transfected into Jurkat cells mediated by liposome, and the cells stably expressing exogenous HCAP1 were screened with G418. The effects of HCAP1 products on cell proliferation were assessed by viable cell count, cell growth curve and colony formation assay in soft agar. The results showed that the HCAP1 transgenic Jurkat cells displayed slow growth rate, extended doubling time and reduced colony formation capability, as compared with the cells transfected with pBK/CMV empty vector (P < 0.01). It is concluded that exogenous HCAP1 gene products could inhibit the proliferation of Jurkat cells.
Carcinoma, Hepatocellular ; genetics ; Cell Division ; Humans ; Jurkat Cells ; Liver Neoplasms ; genetics ; Neoplasm Proteins ; genetics ; Peptides ; Transfection

OBJECTIVEIn order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors.

METHODSA dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining.

RESULTSOne hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05).

CONCLUSIONC8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; Cytoplasm ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hybridomas ; immunology ; secretion ; Male ; Membrane Proteins ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis ; Sphingosine N-Acyltransferase ; Tumor Suppressor Proteins ; immunology ; metabolism

AIMTo explore proper cryopreservative systems for hematopoietic stem cells.

METHODSPeripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation.

RESULTSThe recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group.

CONCLUSION5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

Blood Preservation ; methods ; Cell Survival ; drug effects ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans

OBJECTIVETo sum up the clinical experience of the diagnosis and treatment of intracerebral infiltration by monoclonal plasmacytoid cells in Waldenstrom's macroglobulinemia(Bing-Neel syndrome).

METHODSThe clinical data of the diagnosis and treatment of a case of Bing-Neel syndrome was analyzed.

RESULTSA 56-year-old male was diagnosed as Waldenstrom's macroglobulinemia one year ago, and presented with persistent headache during the treatment period. Magnetic resonance imaging showed a high intensity area on T2-weighed images in the right frontal lobe which was well enhanced by gadolinium-diethylenetriaminepenta-acetic acid. Infiltration of neoplastic cells was confirmed by biopsy. Immunohistochemical examination showed that mature plasmacytoid cells in the cerebral parenchyma were immunoglobulin M positive.

CONCLUSIONInfiltration in CNS (Bing-Neel syndrome) is uncommon in Waldenstrom's macroglobulinemia. As there is no effective therapy for this Bing-Neel syndrome, combination of radiation and chemotherapy should be considered for this situation.

Brain ; pathology ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; Waldenstrom Macroglobulinemia ; pathology