Objective:To obtain a high specificity and high affinity anti-PcrV protein monoclonal antibody which can be used for the treatment of Pseudomonas aeruginosa infected.Methods: The PcrV gene was amplified by PCR using P.aeruginosa PAO1 genome DNA as the template.The expression vector(pET-28a-PcrV) was constructed and transformed into E.coli BL21(DE3).The re-combinant PcrV protein was expressed by IPTG induction and purified by Ni2+affinity chromatography.The specific binders of PcrV were screened by phage display.The genes encoding VH and VL were amplified respectively by PCR using the plasmid of positive clone as the template.Then the recombinant expression vectors were constructed and transfected into 293E cells.Monoclonal antibody were purified by the Protein A affinity resin from the culture supernatants.The affinity of antibody was detected by ELISA and the function of YG5 was verified in murine pneumonia model caused by P.aeruginosa.Results: Recombinant PcrV protein was expressed and purified.A full human monoclonal antibody(named as YG5) against PcrV was obtained by phage display.The results of ELISA showed that YG5 had a high affinity with EC50=61 ng/ml.Furthermore,it was found that YG5 could protect mice from infection caused by P.aeruginosa.Conclusion:Our findings present a novel human monoclonal antibody YG5 against PcrV,which inhibits the infection casued by P.aeruginosa and may be a potential drug for treatment of P.aeruginosa infection.

UPLC-QTOF-MS/MS was used to identify metabolites in rat blood, urine and feces after the administration of n-butanol extract derived from steamed notoginseng. The metabolic process of saponins came from steamed notoginseng was analyzed. The metabolites were processed by PeakView software, and identified according to the structural characteristics of prototype compounds and the accurate qualitative and quantitative changes of common metabolic pathways. Four saponins metabolites were identified based on MS/MS information of metabolites, namely ginsenoside Rh₄, Rk₃, Rk₁, Rg₅,and their 15 metabolites were verified. The metabolic pathways of the four ginsenosides in n-butanol extract included glucuronidation, desugar, sulfation, dehydromethylation, and branch loss. The metabolites of main active saponin components derived from steamed Panax notoginseng were analyzed from the perspective of qualitative analysis. And the material basis for the efficacy of steamed notoginseng was further clarified.

The composition and potency of the high temperature (40℃) stress induced size variants of a recombinant humanized monoclonal antibody (rhumAb1) were characterized by means of SEC-HPLC, nonreduced CE-SDS, liquid chromatography coupled with mass spectrometry (LC-MS) and antibody dependent cell-mediated cytotoxicity (ADCC) assay. The molecular masses of the four size variants (SEC-1-SEC-4) separated by SEC-HPLC and seven size variants (NR-1-NR-7) detected by non-reduced CE-SDS were all characterized by LC-MS. The major low molecular weight variants were generated due to the hinge region fragmentation of heavy chain. The hinge region cleavage was found mainly in the Ser221-Cys-Asp-Lys-Thr-His-Thr-Cys228 sequence, in which C222-D223 and H226-T227 were the major cleavage sites. The size variants of rhumAb1, namely dimer and fragments, have significantly reduced ADCC activity in comparison with the intact rhumAb1 drug product. This study provided insights into the stability profiling for rhumAb1 drug product. The study protocols presented here may be applicable to the analytical characterization of other monoclonal antibody-based therapeutic products.

OBJECTIVETo observe antisepsis, anti-swelling, and therapeutic effects of Fuxiye (FXY), a Chinese medical lotion for external wash in treating vaginitis model rats.

METHODSThe cervicitis rat model was induced by agar plate diffusion, ear auricle swelling induced by dimethylbenzene, and chemical stimulus. The in vitro antibiotic actions of FXY were observed. Besides, its effects on the swelling and inflammation in model rats were also observed.

RESULTSFXY at 25 mg/mL could completely inhibit the growth of Pseudomonas aeruginosa, Escherichia coli, pyogenic Streptococcus, and Streptococcus agalactiae. FXY at 50 mg/mL could completely inhibit the growth of Staphylococcus aureus and Candida albicans. It obviously restrained dimethylbenzene induced ear auricle swelling. It significantly alleviated cervicitis induced by chemical stiumli.

CONCLUSIONFXY showed better effects on antisepsis, anti-inflammation, and treating cervicitis.

Animals ; Anti-Infective Agents ; administration & dosage ; pharmacology ; Anti-Inflammatory Agents ; administration & dosage ; pharmacology ; Dosage Forms ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Rats ; Rats, Sprague-Dawley ; Uterine Cervicitis ; drug therapy ; Vaginitis ; drug therapy

This study was aimed to explore the influence of excessive complement activation on the pathological process of acute graft-versus-host disease (aGVHD) in mice. A murine model with aGVHD was established by injecting cell mixture containing splenocytes and bone marrow cells at 2:1 ratio from donor C57BL/6(H-2K(b)) mice into recipient BALB/c (H-2K(d)) mice within 4-6 hours after 8 Gy (60)Co γ-ray total body irradiation. The mice received syngeneic bone marrow transplantation were used as control group. After transplantation, the mice were monitored daily for body weight and mortality. At day 14, all mice were sacrificed and each liver was freshly dissociated for histological analysis. The hepatic mRNA abundance for complement components C3a and C5a as well as receptors for these two anaphylatoxin were tested by real-time quantitative PCR method. And the levels of C3a and C5a production in liver were detected by ELISA. The deposition of complement C3 in liver was determined by immunofluorescence staining using frozen section. The results indicated that as compared with syngeneic bone-marrow transplantation control group, experimental animals underwent aGVHD characterized by weight loss, depilation, diarrhea and lassitude. Interestingly, the hepatic mRNA expression for complement anaphylatoxin family member C3a and C5a as well as their receptors C3aR and C5aR1 in mice with aGVHD were significantly up-regulated in comparison with control group (p < 0.05). Consistently, the content of C3a and C5a in liver increased markedly in mice with aGVHD (p < 0.01). For animals ongoing aGVHD, complement component C3 depositions were observed in hepatic portal areas, around which massive inflammatory cell infiltration was also observed. It is concluded that in aGVHD animals, excessive complement activation occurs, and the activated complement components participate in pathological process of the aGVHD.
Animals ; Bone Marrow Transplantation ; Complement Activation ; Female ; Graft vs Host Disease ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL

After treating with chemotherapy or immunosuppressant, malignant diseases of hematopoietic system such as leukemia, malignant lymphoma and aplastic anemia usually induced severe infection such as sepsis. Sepsis which is hard to be diagnosed causes high death rate. This study was purposed to establish an experimental sepsis mouse model so as to provide a basis for pathogenesis and intervention study. A classic caecal ligation and puncture (CLP) was used to establish experimental sepsis model. ELISA was used to detect levels of C5a, IL-6, TNFalpha, and IFN-gamma. Flow Cytometry was applied to measure apoptosis of lymphocytes in thymus and mesentery. The pathologic changes of thymus and spleen were confirmed by HE staining. The results showed that almost 70%-80% mice died at 72 hours after CLP. Only approximate 20% animal survived during finite time, mice in CLP group had significant weight lose. Meanwhile large release of different inflammatory mediators which are related with sepsis (C5a, IL-6, TNF-alpha, and IFN-gamma) was observed after CLP. Apoptosis of lymphocytes in thymus and mesentery lymphonodus was enhanced markedly after CLP. Significantly pathologic injury was also observed in thymus and spleen. It is concluded that a mouse model of experimental sepsis was successfully established by caecal ligation and puncture which can well mimic the clinical symptom of sepsis. The experimental sepsis mouse model provides an excellent tool for exploring the pathogenesis and intervention ways for sepsis accompanied with complicated malignant hematological diseases in vivo.
Animals ; Apoptosis ; Cecum ; injuries ; Complement C5a ; metabolism ; Disease Models, Animal ; Interferon-gamma ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sepsis ; metabolism ; pathology ; Spleen ; pathology ; Thymus Gland ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

Objective To study the inhibitory effect of pterin acid (PTA) against ricin and recombinant ricin A chain protein. Methods Luciferase protein synthesis inhibition assay in a cell-free system and in vitro cytotoxicity experiments were performed to assess the biological activity of ricin and rRTA treated with PTA.Results The result showed that PTA could significantly inhibit the activity of ricin and rRTA in a dose-dependent manner.Conclusion PTA might be used as a small molecular probe to develop an evaluating system for ricin/RTA small molecular inhibitor in vitro. The cell-free system adopted in the current study could also serve as a necessary basis for screening some novel small molecular compounds against ricin and RTA in the future.

B-Cell Activating Factor ; antagonists & inhibitors ; metabolism ; B-Lymphocytes ; drug effects ; immunology ; metabolism ; Humans ; Lymphoma, B-Cell ; drug therapy ; immunology ; metabolism ; Models, Biological ; Multiple Myeloma ; drug therapy ; immunology ; metabolism ; Protein Binding ; Receptors, Tumor Necrosis Factor ; antagonists & inhibitors ; metabolism

This study was aimed to investigate the effects of xenogeneic antigen neu-Fc in combination with the recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and Bacillus Calmette-Guerin (BCG) on the regulation of Th1 and Th2 immune response in vitro. The rat neu L2-S2 domain was engineered as a chimeric protein with human IgG Fc. The eukaryotic expression vector was constructed. The recombinant protein was stably expressed in CHO cells and purified by rProtein A Sepharose Fast Flow column. The recombinant protein was identified by SDS-PAGE and Western blot. Peripheral blood mononuclear cells (PBMNCs) were obtained by means of standard Ficoll separation from the blood of healthy donors. Neu-Fc-induced PBMNC proliferation was tested by MTT. The production of IL-12 and IL-10 was measured by ELISA. The results showed that the level of IL-12 decreased and IL-10 increased after PBMNCs were incubated with MCF-7 cultural supernatant. 10 nmol/L neu-Fc strongly induced the cell proliferation. Compared with neu-Fc or GM-CSF or BCG treatment alone, neu-Fc in combination with GM-CSF and BCG significantly stimulated IL-12 production and inhibited IL-10 production (p < 0.01). It is concluded that the neu-Fc can stimulate the proliferation activity of PBMNCs. neu-Fc, GM-CSF and BCG costimulation efficiently induces Th1 immune response.
Animals ; BCG Vaccine ; immunology ; CHO Cells ; Cricetinae ; Cricetulus ; Granulocyte-Macrophage Colony-Stimulating Factor ; immunology ; Humans ; Interleukin-10 ; metabolism ; Interleukin-12 ; metabolism ; Rats ; Recombinant Proteins ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology

AIMTo explore the effects of tRNA on the growth of mammalian cells.

METHODSL929, NIH3T3, MCF-7 and PC12 cells were seeded in 96 well culture plate individually, and incubated at 37 degrees C in 5% CO2 for 4 h, the tRNAs from different species were added to the culture media individually. After certain time of incubation, the viability of the cells was evaluated by the MTT methods. Sub-confluent L929 cells were incubated with 200 microg/ml ytRNA for different times, then the cells were pooled and analyzed with flow cytometry assay.

RESULTStRNA specifically inhibited the growth of L929 cells in a dose-dependent manner. The sizes of tRNA-treated cells showed larger sizes and longer processes than those of untreated cells. Flow cytometric analysis further showed that most of tRNA-treated cells were arrested in S phase of the cell cycle.

CONCLUSIONThe cell growth inhibitory effects of tRNAs were caused mainly by their degraded fragments. The results suggested that tRNA or its degraded fragments might play important roles in regulation of cell proliferation.

Animals ; Cell Cycle Checkpoints ; physiology ; Cell Line ; Cell Proliferation ; Fibroblasts ; cytology ; Flow Cytometry ; Mice ; RNA, Transfer ; physiology