Objective To investigate the change of wave-front aberrations after Ortho-K contact lens(CL)by day and overnight wear.Design Retrospective case series.Participant 180 eyes of 92 myopic patients wearing Ortho-K CL.Method Wave-front aberra- tions of 180 eyes of 92 myopic patients wearing Ortho-K CL were analyzed.Patients were divided into by day wear and overnight wear groups.Wave-front aberrations were measured with WFA 1000B subjective aberrometer before and 1 month after Ortho-K CL wear. Wave-front aberrations at initial,after one month of CL fitting and without CL were measured in same condition,and RMS values for overall wave-front aberrations and each order of the Zernike aberrations were analyzed with Matlab software.Main Outcome Measures RMS value of Zernike coefficient of each order.Result Compared with initial aberration in wear by day group,total RMS and each or- der aberrations(except 2nd order)with lens fitting significantly increased(P0.05).Compared with initial aberration in overnight wear group,overall wavefront aberrations and the RMS(except 2nd,3rd and 6th order Zernike aberrations)with lens fitting significantly increased(P0.05).Conclusion The high order aberrations with lens fitting and without lens fit- ting increased in each group.The aberration with lens fitting in wear by day group was higher than that in overnight wear group,while the aberration without lens fitting in overnight wear group was higher than that in wear by day group.(Ophthalmol CHN,2007,16: 351-354)

Objective To investigate the effects of rigid gas permeable contact lens (RGPCL) wearing 3 years on ocular surface in keratoconus.Design Retrospective case series.Participants 73 patients with keratoconus.Methods From July 2001 to July 2004,73 patients (142 eyes) wearing RGPCL for more than 3 years were collected in Peking University Optometry & Ophthalmology Center.Be- fore and at 1 year,2 years and 3 years of RGPCL wearing,density and morphologic changes of corneal endothelium were examined with non-contact specular microscope.Eye axial length,central and peripheral thickness of cornea were measured with A-scan pachymeter. All eyes were examined with slit-lamp microscope periodically.Main Outcome Measures Corneal endothelial density and morphology, corneal thickness,eye axial length,ocular surface changes.Results Before and at 1 year,2 years and 3 years of RGPCL wearing,aver- age corneal endothelial density was 2901.92?445.20,2862.78?497.13,2854.71?526.80,3015.61?421.22 (cells/mm~2)without significant difference statistically (F=1.571,P=0.20).Morphologic changes were not significant during 3 years.Eye axial length was 25.15?1.50, 24.93?1.36,24.78?1.25,25.39?1.31 (mm) without significant difference statistically (F=2.218,P=0.10).Corneal central thickness was 489.09?59.64,484.02?60.80,496.61?59.74,487.44?54.25(?m)without significant difference statistically (F=0.991,P=0.40).Peripheral thickness changes were also not significant statistically during 3 years.69 eyes with mild conjunctival congestion,12 eyes with corneal fluorescence stain and 6 eyes with corneal epithelial rough were found with slit-lamp microscope during follow-up.Conclusions For the patients with keratoconus,long term of RGPCL wear will not lead to significant ocular changes or severe ocular complications.

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

P-selectin, one of the membrane proteins, expresses on platelet and endothelia and interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocyte membrane. This interaction mediates leukocytes rolling on endothelial membrane and then induces leukocyte recruitment to the site of infection or tissue injury. In the present study, we constructed the recombinant wild type human P-selectin, its calcium-binding sites mutants and recombinant PSGL-1-globulin (PSGL-1-Rg). They expressed in Sf9 cells by using the baculovirus expression system and were purified by TalonTM metal or Protein A affinity chromatography. The results showed that the recombinant PSGL-1-Rg interacted with recombinant wild type P-selectin and two P-selectin mutants with 2 calcium-binding sites mutation respectively, but could not bind to the P-selectin mutant with all 4 calcium-binding sites mutation. Therefore, we verified the importance of P-selectin calcium-binding sites for its interaction with PSGL-1.
Binding Sites ; Calcium ; metabolism ; Humans ; Leukocytes ; metabolism ; Membrane Glycoproteins ; metabolism ; Mutation ; P-Selectin ; metabolism ; Recombinant Proteins ; metabolism

Objective： This study was designed to observe the efficacy and side effects of autologous hematopoietic stem cell transplantation (AHSCT) in the treatment of hematological malignancies and solid tumors. Methods： In the 7 patients, two received autologous bone marrow transplantation (ABMT) and five received autologous peripheral blood stem cell transplantation (APBSCT). The conditioning regimen consisted of VCCA (Vincristine, Me-CCNU, Ara-C, CTX) and TLI(total lymph irradiation) for malignant lymphoma, VCCED (VCR, Me-CCNU, CTX, Vp-16, DNR) and TBI for acute myeloid leukemia-M4, VCCME (VCR, Me-CCNU, CTX, Mitoxantron,Vp-16) for breast cancer. All harvests of stem cells were stored at 4℃ except one at -80℃ ,than transfused back to the patients within 72 hours without purgation. Results： The number of mononuclear cells and CD34＋ cells in harvest in APBSCT patients was (7.45± 5.89)× 108/kg and (18.62± 4.74)× 106/kg,respectively. The patients' granulocyte count reached ≥ 0.5× 109/L on day＋ 9.6 and＋ 11, platelet count reached ≥ 50× 109/L on day ＋ 12.6 and＋ 21.5 and reticulocyte reached ≥ 0.5％ on day＋ 11.2 and＋ 16.5,respectively,after APBSCT and ABMT. The side effects were mainly the gastrointestinal reactions. Up to now, no relapse were seen but four women had developed secondary amenorrhea after AHSCT. Conclusion： AHSCT is an efficient way for malignant tumors and there were earlier hematopoietic reconstitution and fewer side effects in patient with APBSCT than with ABMT. The impairment of gonad by conditioning may affect the quality of life.

We used metabolomics to investigate the ability of a traditional Mongolian medicine called modified Tabusen-2 (MT-2) to improve kidney yang deficiency (KYD) in rats. All animal experiments were conducted under the guidance and standards of the Medical Ethics Committee of Inner Mongolia Medical University. SD rats were divided into 6 groups of six rats: a normal group, a model group, Jinkuishenqi pill administration group (1.26 g·kg^-1), and MT-2 administration in high-, medium- and low-dose groups (1.512, 0.756, and 0.378 g·kg^-1). KYD was established by intramuscular injection of hydrocortisone (HC) and biochemical indicators and clinical characterization was used to confirm that KYD was established. All groups received intragastrically administered drug (Jinkuishenqi pill or MT-2) or saline. Serum from each group was collected after 8 weeks and analyzed by UPLC-Q-exactive-MS to measure various biochemical indicators. The biomarkers affected by MT-2 were identified and the metabolic pathways of KYD regulated by MT-2 were analyzed by metabolomic analysis. The results show that MT-2 can decrease serum creatinine (Cr) in KYD rats and significantly increase (P<0.05) the content of thyroid stimulating hormone (TSH) and luteinizing hormone (LH). In serum samples, 38 biomarkers such as corticosterone, L-phenylalanine, and DL-tryptophan were measured as possible indicators for disease development in KYD rats. MT-2 lowered 18 biomarkers of KYD, including corticosterone, deoxycorticosterone, and L-phenylalanine, and altered 13 related metabolic pathways including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and steroid hormone biosynthesis, resulting in an overall improvement in KYD. MT-2 appears to be important in improving KYD in rats mainly by regulating metabolites such as amino acids, steroids and lipids.

Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Animals ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Transferrin ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; metabolism

BACKGROUNDCytosine deaminase (CD) converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in CD/5-FC gene therapy, 5-FU will be mostly converted into nontoxic beta-alanine without uracil phosphoribosyltransferase (UPRT). UPRT catalyzes the conversion of 5-FU to 5-fluorouridine monophosphate, which directly kills CD::UPRT-expressing cells and surrounding cells via the bystander effect. But the pharmacokinetics and the bystander effect of CD::UPRT/5-FC has not been verified in vivo and in vitro. Before the CD::UPRT/5-FC bi-gene therapy system is used in clinical trial, it is essential to monitor the transgene expression and function in vivo. Thus, we developed a preclinical tumor model to investigate the feasibility of using (19)F-magnetic resonance spectroscopy ((19)F-MRS) and optical imaging to measure non-invasive CD and UPRT expression and its bystander effect.

METHODSC6 and C6-CD::UPRT cells were cultured with 5-FC. The medium, cells and their mixture were analyzed by (19)F-MRS. Rats with intracranial xenografted encephalic C6-CD::UPRT glioma were injected intraperitoneally with 5-FC and their (19)F-MRS spectra recorded. Then the pharmacokinetics of 5-FC was proved. Mixtures of C6 and C6-CD::UPRT cells at different ratios were cultured with 5-FC and the cytotoxic efficacy and survival rate of cells recorded. To determine the mechanism of the bystander effect, the culture media from cell comprising 25% and 75% C6-CD::UPRT cells were examined by (19)F-MRS. A comparative study of mean was performed using analysis of variance (ANOVA).

RESULTS(19)F-MRS on samples from C6-CD::UPRT cells cultured with 5-FC showed three broad resonance signals corresponding to 5-FC, 5-FU and fluorinated nucleotides (F-Nuctd). For the C6 mixture, only the 5-FC peak was detected. In vivo serial (19)F-MRS spectra showed a strong 5-FC peak and a weak 5-FU peak at 20 minutes after 5-FC injection. The 5-FU concentration reached a maximum at about 50 minutes. The F-Nuctd signal appeared after about 1 hour, reached a maximum at around 160 minutes, and was detectable for several hours. At a 10% ratio of C6-CD::UPRT cells, the survival rate was (79.55 +/- 0.88)% (P < 0.01). As the C6-CD::UPRT ratio increased, the survival rate of the cells decreased. (19)F-MRS showed that the signals for 5-FU and F-Nuctd in the culture medium increased as the ratio of C6-CD::UPRT in the mixture increased.

CONCLUSIONS(19)F-MRS studies indicated that C6-CD::UPRT cells could effectively express CD and UPRT enzymes. The CD::UPRT/5-FC system showed an obvious bystander effect. This study demonstrated that CD::UPRT/5-FC gene therapy is suitable for 5-FC to F-Nuctd metabolism; and (19)F-MRS can monitor transferred CD::UPRT gene expression and catalysis of substrates noninvasively, dynamically and quantitatively.

Animals ; Antimetabolites ; pharmacokinetics ; therapeutic use ; Cell Line ; Cytosine Deaminase ; genetics ; physiology ; Flucytosine ; pharmacokinetics ; therapeutic use ; Genetic Therapy ; methods ; Glioma ; drug therapy ; therapy ; Humans ; Magnetic Resonance Imaging ; Male ; Pentosyltransferases ; genetics ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the method of constructing tissue-engineered skin using melanocytes and bone marrow mesenchymal stem cells (BMSCs) in vivo.

METHODSMelanocytes were isolated from human foreskin. BMSCs were isolated from human bone marrow. Both of them were co-cultured at a ratio of 1:10, and then were implanted into the collagen membrane to construct the tissue-engineered skin, which was applied for wound repair in nude mice. The effectiveness of wound repair and the distribution of melanocytes were evaluated by morphological observation, in vivo 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) fluorescent staining tracing, HE staining, S-100 immunohistochemistry, and transmission electron microscopy.

RESULTSThe wounds were satisfactorily repaired among the nude mice. The melanocytes were distributed in the skin with normal structure, as confirmed by DAPI fluorescent staining tracing, HE staining, S-100 immunohistochemistry, and transmission electron microscopy.

CONCLUSIONMelanocytes and BMSCs, after proper in vitro culture at an appropriate ratio, can construct the tissue-engineered skin with I type collagen membrane.

Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Collagen Type I ; Humans ; Melanocytes ; cytology ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Nude ; Skin ; injuries ; Skin, Artificial ; Tissue Engineering

OBJECTIVETo investigate the effect of SiO2 on the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in human silicotic alveolar macrophages (AM).

METHODSHuman alveolar macrophages were collected from a silicotic patient by bronchoalveolar lavage and exposed to silicon dioxide for 3 h, 6 h, 12 h, 18 h, 24 h and 36 h. The expression of the MMP-9 in the AM were detected with zymography and immunological method and the expression of the TIMP-1 in the AM with immunological method.

RESULTSThe expressions of MMP-9 in the AM increased clearly along with the time, reached peak at 24 h when detected with zymography (average optical density: 3.061+/-0.153 vs 2.851+/-0.164, P<0.05); and reached peak at 18h when detected with immunological method (average optical density: 0.386+/-0.037 vs 0.322+/-0.034, P<0.05). The expression of the TIMP-1 in the AM did not vary when detected with immunological method (P>0.05).

CONCLUSIONSiO2 may affect the expression of MMP-9 and MMP-9 activity in the cultured AM.

Cells, Cultured ; Humans ; Macrophages, Alveolar ; drug effects ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Silicon Dioxide ; toxicity ; Silicosis ; pathology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism