This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Chitosan ; administration & dosage ; immunology ; Dendritic Cells ; immunology ; virology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; genetics ; immunology ; Humans ; Immunity, Cellular ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; administration & dosage ; Spleen ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

OBJECTIVETo investigate the potential role of Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in regulation of gene expression of high mobility group box-1 protein (HMGB1) in various tissues in rats with sepsis.

METHODSA sepsis model reproduced by cecal ligation and puncture (CLP), and 128 male Wistar rats were randomly divided into normal control group (n = 10), sham operation group (n = 10), CLP group (n = 60), AG490 treatment group (n = 24), and rapamycin (RPM) treatment group (n = 24). At serial time points animals in each group were sacrificed after CLP, then tissue samples were harvested to determine HMGB1 mRNA expression and STAT1/3 DNA binding activity.

RESULTSSTAT1 activities increased rapidly in the liver, lungs and small intestine after CLP, peaking at 6 - 12 h, while it increased slowly, and still kept at mild level from 2 to 48 h in the kidneys. Compared with STAT1, lower STAT3 activities were detected only in the liver and lungs, with negative detection in the small intestine and kidneys. HMGB1 mRNA levels significantly increased in liver, lungs and small intestine at various time points after CLP respectively (P < 0.05 or P < 0.01), while they didn't change in the kidneys. Treatment with AG490 could markedly inhibit HMGB1 mRNA expression in the liver and small intestine at 24 and 48 h (P < 0.05 or P < 0.01), and in lungs at 2 h following CLP (P < 0.01). Similarly, treatment with RPM significantly decreased HMGB1 mRNA expression in the lungs at 2, 6, 24 and 48 h, in the liver at 6 and 24 h, and in the small intestine at 24 and 48 h (P < 0.05 or P < 0.01). In addition, STAT1 and STAT3 activities in the liver and lungs were significantly correlated with corresponding tissue HMGB1 mRNA expression.

CONCLUSIONSPeritoneal infection could extensively activate STAT1 and limitedly activate STAT3 in vital organs. Activation of JAK/STAT pathway might be involved in up-regulating the gene expression of HMGB1 and systemic inflammation secondary to severe septic challenge.

Animals ; Disease Models, Animal ; Gene Expression ; physiology ; HMGB1 Protein ; genetics ; Janus Kinases ; metabolism ; Male ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; STAT Transcription Factors ; metabolism ; Sepsis ; genetics ; physiopathology ; Signal Transduction

OBJECTIVEThis study aimed to explore the impact of specimen collection and storage consumable products on trace element quantitative analysis.

METHODSDevices and consumable products of different brands used in specimen collection or storage were selected and treated separately as below:urine collection and storage tubes (Brand A, B, C and D, 2 samples for each brand) were treated with 1% of HNO(3) volume fraction for 2 - 4 h; blood taking device (Brand O, P and Q, 3 samples for each brand) were used for ultra-pure water samples collecting as simulation of blood sampling;dust sampling filters (Brand X, Y and Z, 2 samples for each brand) were cold digested by nitric acid for 12 h, followed by microwave digestion. Then cadmium, cobalt, chromium, copper, iron, manganese, molybdenum, nickel, lead, selenium, stannum, titanium, vanadium and zinc concentrations in the solutions obtained during the course of collect or storage were quantified by inductively coupled plasma mass spectrometer.

RESULTSFor the urine collection and storage consumable products, background values of elements were described as mean of parellel samples. The consentration of 14 quantified elements were relatively low for 5 ml cryogenic vials (brand B) with background values range of 0.001 - 0.350 ng/ml. The background values of copper of 50 ml centrifuge tubes (brand A), chromium of 5 ml cryogenic vials (brand C) and zinc of 1.5 ml centrifuge tubes (brand D) were relatively high, which were 1.900, 1.095 and 1.368 ng/ml, respectively. Background values of elements in blood sampling devices were described as x(-) ± s. Background values of chromium for brand O, P and Q were (0.120 ± 0.017), (0.337 ± 0.093) and (0.360 ± 0.035) ng/ml; for copper were (0.050 ± 0.001), (0.017 ± 0.012) and (0.103 ± 0.015) ng/ml; for lead were (0.057 ± 0.072), (0.183 ± 0.118) and (0.347 ± 0.006) ng/ml; for titanium were (7.883 ± 0.145), (8.863 ± 0.190) and (8.613 ± 0.274) ng/ml; zinc were (2.240 ± 0.573), (42.140 ± 22.756) and (8.850 ± 3.670) ng/ml. There were statistically differences of background values for chromium, copper, lead, titanium and zinc among the above three brands of blood sampling devices (all P values < 0.05). For air sampling filters, background values of elements were described as mean of parellel samples. Background values of chromium and nickel of sampling filters (brand X) were lowest, which were 17.000 and 15.400 ng per piece, respectively; while background values for other elements were relatively high, the quantification of cadmium, cobalt, copper, iron, manganese, molybdenum, lead, selenium, stannum, titanium, vanadium and zinc were 0.250, 0.550, 48.500, 690.000, 25.500, 0.900, 6.500, 10.550, 7.950, 10.500, 0.850, 370.000 ng per piece, respectively. Background values of chromium and nickel of sampling filters (brand Z) were highest, which were 171.000 and 29.850 ng per piece.

CONCLUSIONBackground values of trace elements varied among products of different brands, and the most noticable differences were found in chromium, manganese, nickel, lead, stannum and zinc.

Environmental Monitoring ; methods ; Quality Control ; Specimen Handling ; methods ; Trace Elements ; analysis

This study was purposed to investigate the effect of prostaglandin E2 (PGE2) on proliferation of peripheral blood T lymphocytes, and to evaluate the regulatory role of PGE2 on immunological balance between Th1/Th2 and Tc1/Tc2 lymphocytes. The peripheral blood mononuclear cells (PBMNC) were stimulated by anti-human CD3 monoclonal antibody (mAb) and anti-human CD28 mAb, and were cultured in the presence of different concentration of PGE2 for 120 hours. The proliferation of peripheral blood T lymphocytes was assayed according to the manufacture protocol of BrdU Kit; the IFN-gamma and IL-4 levels in supernatants cultured for 24, 48, 72 and 120 hours were detected by ELISA; the ratios of CD4+IL-4+ T cells/CD4+ IFN-gamma+ T cells and CD8+IL-4+ T cell/CD8+IFN-gamma+ T cells were determined by flow cytometry. The cells cultured without PGE2 were used as control. The results indicated that (1) with the raising of concentration of PGE2, the inhibitory rate of T cell proliferation in vitro significantly increased (p=0.001). There was significant positive correlation between inhibitory rate of T cells and PGE2 concentration (correlation coefficient=0.889, p=0.000). (2) the difference between the IFN-gamma concentrations in supernatant cultured for 120 and 72 hours in test groups had no statistical significance (p=0.917). The IFN-gamma concentration increased continually with prolonging of culture time in control group (p=0.046). The IFN-gamma concentrations produced at different times in test group were significantly lower compared with those in control group (p<0.05). The IL-4 concentrations produced at different time had no significant change in test groups (p=0.400). The IL-4 concentration in 24 hours in control group was significantly higher than that at 48, 72 and 120 hours in control group (p=0.007, 0.003 and 0.002). After cultured for 24 hours the IL-4 concentration in test group was significantly lower than that in control group (p=0.037), but after cultured for 48, 72 and 120 hours, the IL-4 concentration in test group did not show statistical difference in comparison with control group (p>0.05). (3) the proportions of CD4+IFN-gamma+T cells in test group and in control group had no significant difference (p=0.767). The proportion of CD4+IL-4+T cells in test group was slightly higher than that in control group (p=0.051). The ratio of CD4+IL-4+T cells to CD4+IFN-gamma+ T cells in test group was significantly higher than that in control group (p=0.011). The proportions of CD8+IFN-gamma+ T cells in test group and in control group had no statistical difference (p=0.441). The proportion of CD8+IL-4+T cells in test group was significantly higher than that in control group (p=0.015). The ratio of CD8+IL-4+ T cells to CD8+IFN-gamma+ T cells in test group were obviously higher than that in control group(p=0.038). It is concluded that the PGE2 inhibits the proliferation of T lymphocytes in vitro. PGE2 influences the production of IFN-gamma and IL-4, and significantly influences peak appearance of IFN-gamma produced by T lymphocyte. PGE2 can continuously inhibit the production of IFN-gamma, but its continuous effect on IL-4 is no significant. PGE2 enhances the ratio of CD4+IL-4+T lymphocytes to CD4+IFN-gamma+T lymphocytes and the ratio of CD8+IL-4+T lymphocytes to CD8+IFN-gamma+T lymphocytes, and regulates development of T cells toward Th2/Tc2 cells.
Cell Proliferation ; drug effects ; Dinoprostone ; pharmacology ; Flow Cytometry ; Humans ; Lymphocyte Activation ; drug effects ; Lymphocyte Count ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Th1 Cells ; drug effects ; immunology ; Th2 Cells ; drug effects ; immunology

OBJECTIVETo explore influence factors of dietary supplement used in population aged above 45 years in Beijing.

METHODSUtilizing the data of the survey of dietary supplement (DS) used in Beijing in 2006 was to investigate the influence factors by using multi-factorial logistic regression analysis.

RESULTSAll 2487 samples were included in the analysis. Sex, age, average income of each month for each member of the family, experiences of deficiency of nutrition, attitude to nutrition knowledge, attitude to the need of DS and city-or-rural resident had shown effects on DS using. The percentage of using DS in female was higher than that in male being 19.3% and 11.8% respectively. The percentage grew up as the age became older. The same trend appeared as the economic-status going up, but not as education level change. Use of DS was greater in urban population (16.9%) than in rural population (14.6%). The percentage of using DS in people who had or just been diagnosed as malnutrition/deficiency disease or chronic disease, or those who thought themselves having got these diseases only by their own feeling were higher than those did not.

CONCLUSIONPeople the female, the elder, or those having higher income, having experiences of deficiency of nutrition, are more interested in nutrition knowledge, and more positive in need of DS and those living in rural should be the target population of nutrition education for DS use.

Age Factors ; Aged ; Aged, 80 and over ; China ; Dietary Supplements ; statistics & numerical data ; Female ; Humans ; Male ; Middle Aged ; Nutrition Surveys ; Sex Factors

OBJECTIVETo study the effect of ex vivo expansion on the migration ability and the CXCR4 expression of umbilical cord blood (CB) hematopoietic stem/progenitor cells (HSPC).

METHODSCD(34)(+) cells isolated from fresh CB samples were cultured in a serum-free and stroma-free culture system. On day 7, 10 and 14, CD(34)(+) cells were re-selected from the expanded cells, and the expression of CXCR4 and the transmigration ability of these CD(34)(+) cells were evaluated respectively and compared with those of the precultured fresh CD(34)(+) cells.

RESULTS(1) SDF-1 induced a higher migration percentage of fresh or expanded CB CD(34)(+) cells than that of uninduced ones. (2) Both of the uninduced and SDF-1-induced migrations were slightly reduced in the first week and then much more reduced in the second week expansion (P < 0.05). (3) The number of the CD(34)(+)CXCR4(+) cells were significantly increased during the culture period, but there was a downtrend of CXCR4 expression on CD(34)(+) subset; the expression levels on day 10 and 14 were lower than that on day 0.

CONCLUSIONSThe expanded HSPC would sustain the chemotactic activity during one-week-culture, but with further extended culture time their intrinsic homing potential would be partly impaired.

Antigens, CD34 ; genetics ; metabolism ; Cell Culture Techniques ; Cell Movement ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Male ; Pregnancy

UNLABELLEDOCTOBER: To explore the effects of the glucagon-like peptide 1 (GLP-1) liraglutide on the penile erectile function of rats with diabetic erectile dysfunction (DED) by observing the impact of liraglutide on the expression of eNOS in the corpus cavernosum of diabetic rats.

METHODSWe randomly divided 30 six-week-old male SD rats into a normal control (n = 10) and an experimental group (n = 20) , established models of diabetes mellitus (DM) in the experimental rats, and subdivided them into a DM (n = 8) and a GLP-1 group (n = 8) to receive intramuscular injection of normal saline and liraglutide at 5 mg per kg of the body weight per day, respectively. After 12 weeks of intervention, we obtained the levels of FPG, FINS, TG, TC, HDL-C, LDL-C, testosterone, and IL-6 and the indexes of Homa-IR and Homa-β, detected the expressions of Akt/p-Akt and eNOS/p-eNOS in the corpus cavernosum by Western blot, and compared the erectile function between different groups.

RESULTSThe frequency and rate of penile erection were significantly lower in the DM group than in the GLP-1 and normal control groups (P < 0.05) and also lower in the GLP-1 group than in the normal controls (P < 0.05). Immunofluorescence staining showed the expression of eNOS mainly in the cytoplasm of the cavernosal vessels and sinusoidal endothelial cells, markedly lower in the DM and GLP-1 groups than in the normal rats (P < 0.05), but higher in the GLP-1 than in the DM group (P < 0.05). The level of eNOS/p-eNOS in the penile tissue was significantly decreased in the DM and GLP-1 groups in comparison with the normal controls (P < 0.01 or P < 0.05), while that of p-eNOS was markedly increased in the GLP-1 group as compared with the DM group (P < 0.05). No statistically significant differences were observed in the Akt level among the three groups of animals (P > 0.05). The expression of p-Akt was remarkably reduced in the DM and GLP-1 groups in comparison with the control rats (P < 0.01 or P < 0.05), but higher in the GLP-1 than in the DM group (P < 0.05).

CONCLUSIONGLP-1 can protect the function of endothelial cells in the corpus cavernosum and improve the erectile function of DED rats by regulating the Akt/ eNOS signaling pathway, which indicates that GLP-1 could be an important option for the treatment and prevention of DED.

Animals ; Blotting, Western ; Diabetes Mellitus, Experimental ; Erectile Dysfunction ; drug therapy ; enzymology ; Hypoglycemic Agents ; pharmacology ; Liraglutide ; pharmacology ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Penile Erection ; drug effects ; Penis ; drug effects ; enzymology ; physiopathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood

Objective:To prepare the synaptophysin and chromogranin A monoclonal antibodies with clinical evalua -tion.Methods:The fuse gene (Syn and CgA) was designed and it was constructed on the expression vector pET-28a.Then ,the fusion protein was purified.After protein immunization , cell fusion and screening , the target antibodies were selected .Specificity study and correlation coefficient of Syn and CgA was evaluated by clinical sample comparison validation .Results:By screening,two antibodies 3D9 and 4A12,respectively,for Syn and CgA,were obtained.19 kinds of wax block organization were detected by 3D9,4A12 and control antibody(Leica).The statistical results were analyzed ,the results were in good agreement ,and the correlation coefficients were r=0.9892 and r =0.9939, respectively.Conclusion: This method is prepared to obtain the synaptophysin and chromogranin A antibodies successfully and both can be used for immunohistochemistry .This method can also provide some reference for the study of antibody .

Objective: To evaluate the prognostic significance of early phase full donor chimerism (FDC) after myeloablative allogeneic peripheral blood stem cell transplantation (allo-PBSCT). Methods: The clinical data of 72 hematological patients received myeloablative allo-PBSCT from Feb. 2016 to Jul. 2017 were analyzed retrospectively. The median age was 36.5 years (range 4-59), 44 were males and 28 females. Of the donors, there were 35 HLA matched sibling donors, 27 haploidentical donors and 10 unrelated donors. Polymerase chain reaction amplification of short tandem repeat sequence (PCR-STR) was used to detect donor cell chimerism (DC) rate of recipient bone marrow at one, two and three months after transplantation. Results: The median follow-up was 462 d (range: 47-805 d), 55 cases were still alive, and 45 cases were disease-free survival (DFS) at the end of follow-up. The 2-year overall survival (OS) and DFS were (68.9±7.7)% and (59.5±6.3)%, respectively. A number of 16 cases underwent relapses, with 2-year cumulative incidence of (24.1±5.3)%. The median time of recurrence was 157(32-374) d. Forty cases (55.6%) developed acute graft-versus-host diseases (aGVHD), with median time of 35.5 (13-90) d. Chronic GVHD (cGVHD) occurred in 23 patients (31.9%), with median time of 169 (94-475) d. Univariate analysis found the following factors were not related to OS, DFS or relapse rate (RR), including age, sex, blood type and sex of donor-recipient, occurrence of aGVHD and cGVHD. The OS and DFS in cases reached FDC and no FDC at two months after transplantation were (85.2±6.9)% vs (66.1±7.7)% (P=0.051) and (76.7±7.7)% vs (48.9±8.1)% (P=0.021), respectively. The RR rate in FDC group was lower than that in no FDC group [(16.6±6.8)% vs (30.4±7.8)%, P=0.187, respectively]. Conclusion: The present study confirmed the important value for predicting the prognosis with whether or not the patients reached FDC at the early phase after allo-PBSCT. The OS and DFS in cases with FDC at two months after transplantation were significantly higher than those of no FDC patients.
Adolescent ; Adult ; Child ; Child, Preschool ; Chimerism ; Female ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Middle Aged ; Peripheral Blood Stem Cell Transplantation ; Prognosis ; Retrospective Studies ; Young Adult

Antibody-drug conjugates (ADCs) are one of the most important classes of anticancer therapeutics. Human epidermal growth factor receptor-2 (HER2), which is highly expressed in many types of aggressive cancers including breast and ovarian cancer, has been approved as an ideal target for ADCs. Lidamycin (LDM), developed by Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, is an enediyne-containing antibiotic with potent anti-tumor activity. LDM is a promising payload for ADCs. In the present research, using a special site-directed conjugating technology, we made a novel ADC (607-LDM) with a drug-to-antibody ratio (DAR) of 2 and composed of the anti-HER2 antibody 607 and LDM. The new ADC exhibited potent antitumor activity against human ovarian cancer SKOV3 and breast cancer BT-474 cells. It also induced apoptosis and G2/M arrest. In nude mice with SKOV3 xenografts and a tumor volume of 150-200 mm³, a single intravenous injection 607-LDM at 1 mg·kg^-1 induced tumor growth inhibition of 72.4%, which was significant compared to either LDM (50.6%) or antibody (30.2%) treatment alone, or both in combination (50.1%, P < 0.05). All animal experiments were performed in accord with National Regulations and approved by the Animal Experiments Ethical Committee of College of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. The novel ADC designed in this study, 607-LDM, is a promising candidate for the treatment of HER2-positive cancers.