The screening and identification of internalized nanobody against EpCAM / 药学学报

Epithelial cell adhesion molecule (EpCAM) is a popular target for cancer therapy. In this research, 3 nanobodies with high specificity and endocytosis activity against EpCAM were developed, which provides a basis for the study of immunotoxin based on EpCAM. In our preliminary experiments, we have immunized a camel with EpCAM-Fc antigen and constructed a high-quality phage display library. Seventeen nanobodies with different complementarity determining region (CDR) 3 sequences have been screened after 3 rounds of biopanning by phage display technology. The animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University School of Pharmacy. After purification, 7 nanobodies showed high cell binding activity by fluorescent activated cell sorting (FACS) identification. Furthermore, 3 nanobodies presented high endocytosis activity based on FACS and laser confocal microscopy, which also showed high affinity to EpCAM measured by ForteBio. According to this study, we aimed to provide a novel alternative approach to the EpCAM-targeted therapy and to provide guidance for the study of nanobody based immunotoxins for other targets.

Construction and screening of phage display library for TIM-3 nanobody / 药学学报

Immunotherapy is a new strategy for cancer treatment that has the potential to treat all types of cancer. T cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3) is a key negative regulator of T cell activation. TIM-3 blockage using anti-TIM-3 monoclonal antibody therapy has a great appeal and special advantages. Nanobodies, derived from heavy chain fragment in camelid animals, are now proving clinical values in the development of antibody drugs. In this study, we have immunized camel with TIM-3 antigens and then constructed phage display library. Moreover, 29 nanobodies with different complementarity-determining regions sequences have been screened from the phage display library by phage display technology. In addition, we successfully constructed the cell line stably expressing TIM-3, and screened 10 TIM-3 nanobodies with high specificity and high affinity using flow cytometry. Our study will lay the foundation for the future screening and development of anti-TIM-3 whole humanized functional nanobody.

Intervention with Schistosoma japonicum cysteine protease inhibitor for treatment of lipopolysaccharide-induced sepsis in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of Schistosoma japonicum cysteine protease inhibitor (rSjCystatin) for treatment of lipopolysaccharide (LPS)-induced sepsis in mice.</p><p><b>METHODS</b>After a week of adaptive feeding, 54 BALB/c mice were randomly divided into normal control group (group A), sepsis group (group B), and rSjCystatin intervention group (group C). The mice in group A received an intraperitoneal injection of PBS (100 µL), and those in groups B and C were injected with PBS (100 µL) containing LPS (10 mg/kg); the mice in group C were also intraperitoneally injected with 25 µg sjCystatin in 100 µL PBS 30 min after LPS injection. From each group, 10 mice were randomly selected 24 h after PBS or LPS injection for detecting serum levels of TNF-α, IL-6, and IL-10 using ELISA and the levels of ALT, AST, BUN, and Cr using automatic biochemical analyzer; the pathological changes in the liver, lung and kidney were observed with HE staining. The remaining 8 mice in each group were used for observing the changes in the general condition and the 72-h survival.</p><p><b>RESULTS</b>The 72-h survival rates of the mice was 100% in group A, 0 in group B, and 36% in group C, showing a significant difference among the 3 groups (P<0.05). Compared with those in group A, the mice in group B exhibited obvious liver, lung, and renal pathologies with increased levels of ALT, AST, BUN, Cr, IL-6, and TNF-α (P<0.05). Treatment with sjCystatin significantly lessened LPS-induced organ pathologies, lowered the levels of liver and renal functional indexes and the pro-inflammatory cytokines, and increased the serum level of IL-10 in the mice (P<0.05).</p><p><b>CONCLUSION</b>SjCystatin can produce a significant therapeutic effect on sepsis induced by LPS in mice.</p>

In vitro release and in situ gastrointestinal absorption of evodiamine butyryl derivative-loaded solid lipid nanoparticles / 中国药理学通报

Aim To prepare evodiamine butyryl deriva-tive (EBD) and evodiamine butyryl derivative-loaded solid lipid nanoparticles (EBDLN), and study its re-lease in vitro,and to investigate its in situ gastrointesti-nal absorption. Methods EBD was prepared by a one-step synthetized method, and then EBDLN was prepared by a film dispersion method. Dynamic dialy-sis was used to evaluate drug release in vitro,and sin-gle-pass gastrointestinal perfusion was employed to study the gastrointestinal absorption of EDM,EBD and EBDLN. Results In identical release media, there were identical drug release tendencies of EBD and EB-DLN, but the release rate of EBDLN was faster than EBD. Compared with EDM and EBD, the Kavalues and Pappvalues of EBDLN in every perfusion segment increased significantly. The Kaof EBDLN in stomach, duodenum, jejunum, ileum and colon was 110.14-fold,56.70-fold,51.23-fold,45.70-fold and 127.23-fold of free EDM respectively. The Pappvalue of EB-DLN was 9.74-fold, 4.48-fold, 3.82-fold and 11.3-fold of that of free EDM. Conclusion EBDLN has sustained effect and can enhance the gastrointestinal absorption of EDM and EBD.

Progress in the application of neural oscillations cross-frequency coupling in cognitive function research / 生理学报

Neural oscillations cross-frequency coupling (CFC) refers to the effect of the cross modulation between the electrophysiological oscillation rhythm in different ensembles of neurons. The CFC can reflect the mechanism of information transfer and exchange of local field potentials, electroencephalograph (EEG) and other neural electrophysiological activities at different spatial and temporal scales and plays an important role in the study of cognitive function. This paper introduces the basic phenomenon and classifications of neural oscillation CFC briefly, and reviews the typical applications in the study of the animal cognition model and human cognitive function in recent years, respectively. The main problems are also summarized and the future research is prospected in order to provide new ideas to promote the study and application of the CFC.

Effect of Trichinella spiralis and its worm-derived proteins on CLP-induced sepsis in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of Trichinella spiralis and its worm-derived proteins on cecal ligation and puncture (CLP)-induced sepsis in mice.</p><p><b>METHODS</b>Eighty male BALB/c mice were randomly divided into sham-operated group, CLP group, Trichinella spiralis muscle larvae (ML) pre-infection group (ML+CLP group), soluble muscle larvae proteins (SMP) treatment group (SMP+CLP group) and excretory-secretory proteins (MES) treatment group (MES+CLP group). In ML+CLP group, the mice were orally infected with 300 Trichinella spiralis muscle larvae at 28 days before CLP and those in the other groups were intraperitioneally injected with PBS or SMP (25 µg/mice) or MES (25µg/mice) 30 min after CLP. The general condition and 72-h survival after CLP of the mice were observed. The levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), creatinine (Cr), TNF-α, IL-6, IL-1β, IL-10 and TGF-β were measured at 12 h after the operation, and the pathological changes of the liver and kidney were observed.</p><p><b>RESULTS</b>s Compared with the sham-operated mice, the mice in CLP group showed decreased 72-h survival, obviously increased ALT, AST, BUN, Cr, TNF-α, IL-6, IL-1β, IL-10, and TGF-β with hepatic cords disorder, hepatocytes swelling, glomerulus shrinkage, and renal tubular cell edema. Compared with CLP group, the mice in ML+CLP group showed lowered levels of ALT, AST, Cr, TNF-α and IL-1β and increased levels of IL-10 and TGF-β; in SMP+CLP group, the levels of ALT, AST, Cr, TNF-α and IL-1β were decreased and TGF-β increased. In MES+CLP group, the mice showed obviously increased 72-h survival with lowered levels of ALT, AST, BUN, Cr, TNF-α, IL-6 and IL-1β, increased levels of IL-10 and TGF-β, and alleviated liver and kidney damages.</p><p><b>CONCLUSION</b>Trichinella spiralis and its worm-derived proteins can decrease the levels of pro-inflammatory cytokines and increase immunomodulatory cytokines, and MES has more potent effect to reduce structural and functional damages of the liver and kidney.</p>

Kallikrein-binding protein promotes axonal regeneration and protect rat retinal ganglion cells following optic nerve injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect of kallikrein-binding protein (KPB) in protecting retinal ganglion cells (RGCs) and promoting axonal regeneration following optical nerve injury in rats.</p><p><b>METHODS</b>Crush injury of the optic nerve at 0.5-1.0 mm from the eyeball was induced in rats, which received subsequent KBP injection into the vitreous cavity (experimental group) and PBS injection (control group). At 7, 14 and 21 days after the injury, the rats were sacrificed and frozen sections of the eyeball were prepared to observe the structure and thickness of the retina and count the number of survival RGCs with HE staining. The optic nerves were collected for Western blotting to assess the effect of KBP on the RGCs and axonal regeneration.</p><p><b>RESULTS</b>RGC counts and retinal thickness showed significant differences between the two groups. Western blotting also demonstrated a significant difference in the expression of the nerve regeneration marker protein GAP-43 between the two groups.</p><p><b>CONCLUSION</b>KBP offers protection on RGCs and promotes regeneration of the optic nerve axons after optic nerve injury in rats.</p>

Changes in glial fibrillary acidic protein and growth-associated protein-43 expressions in retinal ganglial cells during axonal regeneration / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the changes in the expressions of glial fibrillary acidic protein (GFAP) and growth- associated protein-43 (GAP-43) in retinal ganglial cells after neural transplantation.</p><p><b>METHODS</b>Thirty-nine rats were randomized into normal control group, nerve amputation group and nerve amputation with peripheral nerve transplantation group. Immunohistochemistry was used to detect the changes in the expressions of GFAP and GAP-43 at different time points after the operations, and real-time PCR was employed to detect the mRNA expressions of 13 genes in the retinal ganglial cells of the rats.</p><p><b>RESULTS</b>Immunohistochemistry showed obviously increased GFAP expressions in the retina following the nerve amputation. GFAP expression was down-regulated while GAP-43 expression upregulated in the retinal ganglial cells after peripheral nerve transplantation. Real-time PCR results showed that 5 days after the operations, retinal GFAP and GAP-43 expressions increased significantly in the nerve amputation group and peripheral nerve transplantation groups as compared with those in the control group, but GAP-43 expression decreased significantly in the former two groups afterwards.</p><p><b>CONCLUSION</b>The regenerated retina may adjust the production of GFAP. The retinal ganglial cells express GAP-43 during retinal regeneration. Up-regulation of the expression of GAP-43 provides the evidence for nerve regeneration following the nerve transplantation.</p>

Properties of the composite membrane of chitosan/nanometer multilayer hydroxyapatite/gelatin / 中国组织工程研究

BACKGROUND:Chitosan and gelatin have good biocompatibility and biodegradable properties.More and more attentions have been given to the application of the composite membrane of chitosan/hydroxyapatite(HA)and gelatin/HA as the tissue scaffolds.OBJECTIVE:To prepare a biocompatible composite membrane of chitosan/HA/gelatin having nanometer multilayer HA,and to investigate whether such a membrane might be a candidate of the tissue scaffolds for the bond repair and regeneration,DESIGN:Controlled observation.SETTING:College of Chemistry and Environmental Engineering,Jianghan UniversityMATERIALS:The experiments were performed at the Laboratory of Fine Chemistry,College of Chemistry and Environmental Engineering,Jianghan University from February to August 2007.Chitosan (Yuhnan Ocean Biochemistry Co.,Ltd,Zhengjiang,China,Mw 13.44×104,93% degree of deacetylation),and gelation(provided by Shanghai Chemical Reagent Co.,Shanghai,China)were used in the present study.METHODS:The mixture chitosan/CaCl2 solution was casted on a glass plate to give the membrane of chitosan/CaCl2.Then,the membrane was soaked in KH2PO4 solution,0.1 mol/L NaOH solution,and 3 wt% gelatin aqueous solution,respectively.The resulted membrane was washed and dried to obtain the chtiosan/HA/gelatin composite membrane.The morphological observation of the composite membrane and HA crystal were carded on a scanning electron microscope.The mechanical properties of the composite membrane were measured using the universal testing machine.The tensile strength(σb)and the elongation at break (ε)were calculated.The thermal stability of composite membrane was determined using a WCT-2C thermobalance.MAIN OUTCOME MEASURES:The morphologies,the mechanical properties,and the thermal stability of the composite membrane and HA crystal were observed.RESULTS:The multilayer HA crystal with 400 nm thickness in the composite membrane was observed.The tensile strength(σb)and the elongation at break(ε)of the composite membrane were 3.83-10.25 MPa and 3.97%-10.14%,respectively.The decomposition temperature of HA-treated membrane was 310℃,which was higher than that of chitosan/gelatin membrane(305℃).CONCLUSION:The composite chitosan/HA/gelatin membrane was prepared by using chitosan/CaCl2,KH2PO4 and gelatin solutions.Such a biocompatible composite membrane with multilayer HA might be a promising tissue scaffold.

Research on the progress of neuroprosthesis for the limb motor system / 中国医疗器械杂志

This paper reviews the current developing situation of neuroprostheses which are based on the functional electrical stimulation (FES) technique and are used to provide limb movements. It also discusses the major challenges of neuroprostheses, which will be faced in the future development and in the clinical applications for the rehabilitation of spinal cord injury and stroke.