Wrist-ankle Acupuncture Increases Pain Thresholds in Healthy Adults.

Context • Wrist-ankle acupuncture (WAA) has been used to relieve both chronic and acute pain in China. Some research has shown that WAA can increase the pain thresholds in pain patients, but the ability of WAA to affect the pain thresholds in healthy adults is unknown. Objective • The study intended to assess the influence of WAA on the pain thresholds of healthy adults. Design • This is an observational study. Setting • This study was conducted in the School of Traditional Chinese Medicine at the Second Military Medical University (Shanghai, China). Participants • Participants were 50 healthy university students aged 19-23 y. Intervention • In the theory of WAA, each side of the body and each limb are longitudinally divided into 6 regions, with 1 needling point defined for each region at the wrist or ankle. The theory indicates that needling a point should relieve pain in a point's corresponding region. For the study, a needle was inserted and retained for 30 min in the Upper 2 point of the left wrist of each participant. Outcome Measures • The pressure pain threshold was measured by a handheld algometer at a position in the left Upper 2 region corresponding to the site of the needling and at positions in the right Upper 2 region as well as the left and right Upper 3 regions not corresponding to the site of the needling. The measurements were taken at 40 min before needling, 5 min after needling, 30 min after needling when the needles were removed, and 70 min after needling. Results • The immediate influence of the WAA on the pain threshold was not significant at 5 min after needling (P > .05). However, at 30 min after needling when the needles were removed, the increases in the pain thresholds were statistically significant when compared to those at 40 min before needling, which were the measurements at baseline (P ≤ .01). At 70 min after needling, the pain thresholds remained higher than those at 40 min before needling (P < .05). From 40 min before needling to 70 min after needling, the pain thresholds in the different positions showed a continuous increase. Conclusions • The WAA had an analgesic effect on pressure-induced pain not only in the corresponding but also in the noncorresponding regions of the needling point in healthy adults. The immediate analgesic effect of the WAA at 5 min after needling was not obvious, but the effects at 30 min and 70 min after needling were statistically significant.

Pharmacokinetics and brain uptake of an IgG-TNF decoy receptor fusion protein following intravenous, intraperitoneal, and subcutaneous administration in mice.

Tumor necrosis factor (TNF)-α is a proinflammatory cytokine active in the brain. Etanercept, the TNF decoy receptor (TNFR), does not cross the blood-brain barrier (BBB). The TNFR was re-engineered for BBB penetration as a fusion protein with a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb domain of the fusion protein acts as a molecular Trojan horse and mediates transport via the endogenous BBB TfR. To support future chronic treatment of mouse models of neural disease with daily administration of the cTfRMAb-TNFR fusion protein, a series of pharmacokinetics and brain uptake studies in the mouse was performed. The cTfRMAb-TNFR fusion protein was radiolabeled and injected into mice via the intravenous, intraperitoneal (IP), or subcutaneous (SQ) routes of administration at doses ranging from 0.35 to 10 mg/kg. The distribution of the fusion protein into plasma following the IP or SQ routes was enhanced by increasing the injection dose from 3 to 10 mg/kg. The fusion protein demonstrated long circulation times with high metabolic stability following the IP or SQ routes of injection. The IP or SQ routes produced concentrations of the cTfRMAb-TNFR fusion protein in the brain that exceed by 20- to 50-fold the concentration of TNFα in pathologic conditions of the brain. The SQ injection is the preferred route of administration, as the level of cTfRMAb fusion protein produced in the brain is comparable to that generated with intravenous injection, and at a much lower plasma area under the concentration curve of the fusion protein as compared to IP administration.

Comparison of registered and published primary outcomes in randomized controlled trials of gastroenterology and hepatology.

OBJECTIVES. The need for trial registration as well as the benefits it has brought for the transparency of medical research has been recognized for years. Trial registration has turned from an exception to a mandatory guideline in recent years. The present study aimed to examine the characteristics of registered randomized controlled trials (RCTs) in a sample of recently published gastroenterology RCTs, and to assess the consistency of registered and published primary outcome (PO) in RCTs. METHODS. Articles published in the top five "general and internal journals" and top five "gastroenterology and hepatology journals" categories between 2009 and 2012 were searched in PubMed. Basic characteristics and the registration information were identified and extracted from the included RCTs. PO consistency analysis was conducted to compare between the registered and published format. RESULTS. A total of 305 RCTs were included; among them 252 could be identified with a registration number. Nearly half of these RCTs were funded solely by industry (141/305, 46.3%). ClinicalTrials.gov was the most popular registry for these RCTs (214/252, 84.9%). A total of 155 RCTs were included in the PO consistency analysis. Among them, 22 (14.2%) RCTs had discrepancies between POs registered in the trial registry compared to the published article. CONCLUSIONS. Based on the results of the present study, selective outcome reporting of gastroenterology RCTs published in leading medical journals has been much improved over the past years. However, there might be a sampling bias to say that consistency of registered and published POs of gastroenterology RCTs has been better than before.

Brain-penetrating IgG-iduronate 2-sulfatase fusion protein for the mouse.

Mucopolysaccharidosis (MPS) type II (Hunter's syndrome) is caused by mutations in the iduronate 2-sulfatase (IDS) fusion protein. MPS-II affects the brain, and enzyme replacement therapy is not effective in the brain, because the enzyme does not cross the blood-brain barrier. To treat mouse models of MPS-II with brain-penetrating IDS, the lysosomal enzyme was reengineered as an IgG-IDS fusion protein. The mature human IDS was fused to the carboxyl terminus of both heavy chains of the chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and the fusion protein is designated cTfRMAb-IDS. The purity and identity of the fusion protein was confirmed by electrophoresis and Western blotting with antibodies to mouse IgG and human IDS. The EC50 of binding of the cTfRMAb-IDS fusion protein to the mouse TfR (0.85 ± 0.15 nM) was comparable to the EC50 of binding of the cTfRMAb (0.78 ± 0.05 nM). The IDS enzyme activity of the cTfRMAb-IDS fusion protein was 126 ± 1 nmol · h⁻¹ · µg⁻¹ protein. After intravenous injection in the mouse, the cTfRMAb-IDS fusion protein was rapidly removed from plasma and distributed to tissues, including brain and spinal cord. The uptake of the fusion protein by brain or spinal cord was 1.3 ± 0.1 and 2.2 ± 0.2% injected dose/g, respectively, which is 100-fold greater than the brain uptake of IDS alone. This work shows that a lysosomal sulfatase can be reengineered as an IgG-enzyme fusion protein that rapidly penetrates the brain after intravenous administration.

Synthesis of bisethylnorspermine lipid prodrug as gene delivery vector targeting polyamine metabolism in breast cancer.

Progress in the development of nonviral gene delivery vectors continues to be hampered by low transfection activity and toxicity. Here we proposed to develop a lipid prodrug based on a polyamine analogue bisethylnorspermine (BSP) that can function dually as gene delivery vector and, after intracellular degradation, as active anticancer agent targeting dysregulated polyamine metabolism. We synthesized a prodrug of BSP (LS-BSP) capable of intracellular release of BSP using thiolytically sensitive dithiobenzyl carbamate linker. Biodegradability of LS-BSP contributed to decreased toxicity compared with nondegradable control L-BSP. BSP showed a strong synergistic enhancement of cytotoxic activity of TNF-related apoptosis-inducing ligand (TRAIL) in human breast cancer cells. Decreased enhancement of TRAIL activity was observed for LS-BSP when compared with BSP. LS-BSP formed complexes with plasmid DNA and mediated transfection activity comparable to DOTAP and L-BSP. Our results show that BSP-based vectors are promising candidates for combination drug/gene delivery.

[Association of MMP14 gene polymorphisms and osteoporosis in Zhuang men from Baise region of Guangxi].

OBJECTIVE: To assess the association between membrane type 1 matrix metalloproteinase gene (MT1-MMP, MMP14) polymorphisms and osteoporosis in Zhuang men from Baise region of Guangxi. METHODS: Genotypes of 5 loci (rs1003349, rs3751488, rs2269213, rs2236303 and rs743257) of MMP14 gene in 301 Zhuang men were determined with single base extension methods, and bone mineral density (BMD) at left calcaneus was evaluated with quantitative ultrasound with measured values of broadband ultrasonic attenuation (BUA). The subjects were divided according to BMD into osteoporosis group, osteopenia group and normal bone density group. RESULTS: All selected loci were in Hardy-Weinberg equilibrium (P> 0.05). By multiple linear stepwise regression analysis, polymorphisms of the five loci were not associated with BUA. But a significant higher risk of osteoporosis was found in individuals with MMP14 rs1003349 GT genotype (vs. GG genotype; P<0.05) and rs2236303 CC and CT genotypes (vs. TT genotype; P<0.05). Genetic linkage between rs1003349 and rs2236303 was also discovered (D'= 0.839, r(2) = 0.458, P<0.01). Compared with the normal bone density group, the frequency of a G-T haplotype of rs1003349 and rs2236303 was significantly lower in the osteoporosis group (P<0.05). And the risk of osteoporosis for individuals with G-C and T-C haplotypes was 2.556 (95% CI: 1.029-6.349, P = 0.038) and 5.111 (95% CI: 1.341-19.485, P = 0.011) compared with G-T haplotype. CONCLUSION: Polymorphisms of rs1003349 and rs2236303 loci of MMP14 gene are associated with the susceptibility of osteoporosis in Zhuang men in Guangxi. G-C and T-C haplotypes for loci rs1003349 and rs2236303 may increase the disease risk.

A traditional Chinese herbal medicine compound preparation versus interventional therapy after resection of small hepatocellular carcinoma: 22-year follow-up.

OBJECTIVE: To compare the long-term effectiveness of compound Ruanjianhugan(RJH)tablets and interventional therapy (IT) in patients after resection of small hepatocellular carcinoma (HCC). in 399 patients after resection of small HCC who were admitted between January 1987 and December 2008 in the Department of Hepatobiliary Surgery and Center of Minimally Invasive Surgery, First Affiliated Hospital of Guangxi Medical University. Four groups were based on different therapy modes: a TCM-only (TCMO) group, a TCM combined with interventional therapy (TCM-IT) group, an interventional therapy-only (ITO) group, and a simple operation (SO) group. Prognostic factors were correlated with overall survival (OS) and OS rates were calculated with the Kaplan-Meier method, and multivariate analyses for factors affecting survival were evaluated by the Cox proportional hazard model. RESULTS: The median OS was 151.20 months in the TCM-IT group, 43.87 months in the ITO group, and 20.77 months in the SO group. All survival rates of the TCMO group were higher than those of the other three groups (>50%). The 5-, 10-, and 15-year OS in the TCMO and ITO patients were 83.94%, 45.50%, and 71.22% and 33.34%, 55.58%, and 9.26%, respectively (risk ratio, 0.209; 95% confidence interval, 0.126-0.347; P = 0.000). Multivariate analysis revealed that the independent risk factors were therapy mode (P = 0.000), sex (P = 0.005), family history (P = 0.011), TNM classification of malignant tumor staging (P = 0.000), medical care-seeking behavior (P = 0.021), and maximum diameter (P = 0.030). CONCLUSION: Long-term oral use of compound RJH tablets may improve OS for small HCC after resection compared with IT.

[Assessment of research papers in medical university staff evaluation].

Medical university staff evaluation is a substantial branch of education administration for medical university. Output number of research papers as a direct index reflecting the achievements in academic research, plays an important role in academic research evaluation. Another index, influence of the research paper, is an indirect index for academic research evaluation. This paper mainly introduced some commonly used indexes in evaluation of academic research papers currently, and analyzed the applicability and limitation of each index. The author regards that academic research evaluation in education administration, which is mainly based on evaluation of academic research papers, should combine the evaluation of journals where the papers are published with peer review of the papers, and integrate qualitative evaluation with quantitative evaluation, for the purpose of setting up an objective academic research evaluation system for medical university staff.

Neuroprotection with a brain-penetrating biologic tumor necrosis factor inhibitor.

Biologic tumor necrosis factor (TNF)-α inhibitors do not cross the blood-brain barrier (BBB). A BBB-penetrating TNF-α inhibitor was engineered by fusion of the extracellular domain of the type II human TNF receptor (TNFR) to the carboxyl terminus of the heavy chain of a mouse/rat chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb-TNFR fusion protein and etanercept bound human TNF-α with high affinity and K(D) values of 374 ± 77 and 280 ± 80 pM, respectively. Neuroprotection in brain in vivo after intravenous administration of the fusion protein was examined in a mouse model of Parkinson's disease. Mice were also treated with saline or a non-BBB-penetrating TNF decoy receptor, etanercept. After intracerebral injection of the nigral-striatal toxin, 6-hydroxydopamine, mice were treated every other day for 3 weeks. Treatment with the cTfRMAb-TNFR fusion protein caused an 83% decrease in apomorphine-induced rotation, a 67% decrease in amphetamine-induced rotation, a 82% increase in vibrissae-elicited forelimb placing, and a 130% increase in striatal tyrosine hydroxylase (TH) enzyme activity. In contrast, chronic treatment with etanercept, which does not cross the BBB, had no effect on neurobehavior or striatal TH enzyme activity. A bridging enzyme-linked immunosorbent assay specific for the cTfRMAb-TNFR fusion protein showed that the immune response generated in the mice was low titer. In conclusion, a biologic TNF inhibitor is neuroprotective after intravenous administration in a mouse model of neurodegeneration, providing that the TNF decoy receptor is reengineered to cross the BBB.

Delivery of a peptide radiopharmaceutical to brain with an IgG-avidin fusion protein.

The genetic engineering, host cell expression, purity, identity, and in vivo brain drug targeting properties are described for a new IgG-fusion protein, designated the cTfRMAb-AV fusion protein. Avidin (AV) is fused to the carboxyl terminus of the heavy chain of the genetically engineered chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR). The TfRMAb binds the endogenous TfR on the blood-brain barrier (BBB), which triggers transport into brain from blood. The cTfRMAb-AV fusion protein is produced in stably transfected Chinese hamster ovary cells, which are grown in serum free medium under conditions of biotin starvation. Following affinity purification, the purity and identity of the cTfRMAb-AV fusion protein were verified by electrophoresis and Western blotting. The affinity of the cTfRMAb for the murine TfR is high, K(I) = 4.6 ± 0.5 nM, despite fusion of avidin to the antibody heavy chain. The model peptide radiopharmaceutical used in this study is the Aß(1-40) amyloid peptide of Alzheimer's disease (AD), which in a brain-penetrating form could be used to image the amyloid plaque in brain in AD. The BBB transport and brain uptake of the [(125)I]-Aß(1-40) peptide was measured in mice injected intravenously (IV) with the peptide either free or conjugated to the cTfRMAb-AV fusion protein. The brain uptake of the free Aß(1-40) peptide was very low, 0.1% of injected dose (ID)/gram brain following i.v. injection, and is comparable to the brain uptake of a brain blood volume marker. However, the brain uptake of the Aß(1-40) peptide was high, 2.1 ± 0.2% ID/gram brain, following attachment of the biotinylated peptide to the cTfRMAb-AV fusion protein. Capillary depletion analysis showed the peptide penetrated the brain parenchyma from blood. The cTfRMAb-AV fusion protein is a new drug delivery system that can target to mouse brain monobiotinylated peptide or antisense radiopharmaceuticals.

Brain-penetrating tumor necrosis factor decoy receptor in the mouse.

Biologic tumor necrosis factor inhibitors (TNFIs) include TNF decoy receptors (TNFRs). TNFα plays a pathologic role in both acute and chronic brain disease. However, biologic TNFIs cannot be developed as brain therapeutics because these large molecule drugs do not cross the blood-brain barrier (BBB). To enable penetration of the brain via receptor-mediated transport, the human TNFR type II was re-engineered as an IgG fusion protein, where the IgG part is a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-TNFR. The cTfRMAb part of the fusion protein acts as a molecular Trojan horse to ferry the TNFR across the BBB via transport on the endogenous BBB TfR. cTfRMAb-TNFR was expressed by stably transfected Chinese hamster ovary cells and purified by affinity chromatography to homogeneity on electrophoretic gels. The fusion protein reacted with antibodies to both mouse IgG and the human TNFR and bound TNFα with high affinity (K(d) = 96 ± 34 pM). cTfRMAb-TNFR was rapidly transported into mouse brain in vivo after intravenous administration, and the brain uptake of the fusion protein was 2.8 ± 0.5% of injected dose per gram of brain, which is >45-fold higher than the brain uptake of an IgG that does not recognize the mouse TfR. This new IgG-TNFR fusion protein can be tested in mouse models of brain diseases in which TNFα plays a pathologic role.

Chronic dosing of mice with a transferrin receptor monoclonal antibody-glial-derived neurotrophic factor fusion protein.

Glial-derived neurotrophic factor (GDNF) is a potential neurotrophic factor treatment of brain disorders, including Parkinson's disease. However, GDNF does not cross the blood-brain barrier (BBB). A brain-penetrating form of GDNF, which is a fusion protein of human GDNF and a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR), has been engineered for the mouse and is designated the cTfRMAb-GDNF fusion protein. The present study examined the potential toxic side effects and immune response after treatment of mice with twice-weekly cTfRMAb-GDNF fusion protein at a dose of 2 mg/kg i.v. for 12 consecutive weeks. Chronic treatment with the fusion protein caused no change in body weight, no change in 23 serum chemistry measurements, and no histologic changes in brain and cerebellum, kidney, liver, spleen, heart, or pancreas. Chronic treatment caused a low-titer immune response against the fusion protein, which was directed against the variable region of the antibody part of the fusion protein, with no immune response directed against either the constant region of the antibody or against GDNF. A pharmacokinetics and brain uptake study was performed at the end of the 12 weeks of treatment. There was no change in clearance of the fusion protein mediated by the TfR in peripheral organs, and there was no change in BBB permeability to the fusion protein mediated by the TfR at the BBB. The study shows no toxic side effects from chronic cTfRMAb-GDNF systemic treatment and the absence of neutralizing antibodies in vivo.

Expression in CHO cells and pharmacokinetics and brain uptake in the Rhesus monkey of an IgG-iduronate-2-sulfatase fusion protein.

Sulfatases are potential therapeutic biopharmaceuticals, as mutations in sulfatase genes leads to inherited disease. Mucopolysaccharidosis (MPS) Type II is caused by mutations in the lysosomal enzyme, iduronate-2-sulfatase (IDS). MPS-II affects the brain and enzyme replacement therapy is ineffective for the brain, because IDS does not cross the blood-brain barrier (BBB). To deliver IDS across the human BBB, the sulfatase has been re-engineered as an IgG-sulfatase fusion protein with a genetically engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb part of the HIRMAb-IDS fusion protein acts as a molecular Trojan horse to ferry the fused IDS across the BBB. Chinese hamster ovary (CHO) cells were stably transfected to produce the HIRMAb-IDS fusion protein. The fusion protein was triaged to the lysosomal compartment of MPS-II fibroblasts based on confocal microscopy, and 300 ng/mL medium concentrations normalized IDS enzyme activity in the cells. The HIRMAb-IDS fusion protein was tritiated and injected intravenously into the adult Rhesus monkey at a low dose of 0.1 mg/kg. The IDS enzyme activity in plasma was elevated 10-fold above the endogenous level, and therapeutic plasma concentrations were generated in vivo. The uptake of the HIRMAb-IDS fusion protein in the brain was sufficiently high to produce therapeutic concentrations of IDS in the brain following IV administration of the fusion protein.

Receptor-mediated abeta amyloid antibody targeting to Alzheimer's disease mouse brain.

The goal of this work is the reduction in the Abeta amyloid peptide burden in brain of Alzheimer's disease (AD) transgenic mice without the concomitant elevation in plasma Abeta amyloid peptide. An anti-Abeta amyloid antibody (AAA) was re-engineered as a fusion protein with a blood-brain barrier (BBB) molecular Trojan horse. The AAA was engineered as a single chain Fv (ScFv) antibody, and the ScFv was fused to the heavy chain of a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this fusion protein was designated cTfRMAb-ScFv. The cTfRMAb-ScFv protein penetrates mouse brain from blood via transport on the BBB TfR, and the brain uptake is 3.5% of injected dose/gram brain following an intravenous administration. Double transgenic APPswe,PSEN1dE9 mice were studied at 12 months of age. The mice were shown to have extensive Abeta amyloid plaques in cerebral cortex based on immunocytochemistry. The mice were treated every 3-4 days by intravenous injections of either saline or the cTfRMAb-ScFv fusion protein at an injection dose of 1 mg/kg for 12 consecutive weeks. The brain A߹⁻4² concentration was reduced 40% in the fusion protein treated mice, without any elevation in plasma A߹⁻4² concentration. No cerebral microhemorrhage was observed in the treated mice. These results show that brain-penetrating antibody pharmaceutics can be developed for brain disorders such as AD following the re-engineering of the antibody as a fusion protein that is transported across the BBB via receptor-mediated transport.

Reversal of lysosomal storage in brain of adult MPS-I mice with intravenous Trojan horse-iduronidase fusion protein.

A mouse model of mucopolysaccharidosis (MPS) type I, which is null for the lysosomal enzyme, α-L-iduronidase (IDUA), is treated with intravenous, receptor-mediated enzyme replacement therapy of the brain. Murine IDUA, which does not cross the blood-brain barrier, is re-engineered for targeting to the brain as an IgG-enzyme fusion protein. The amino terminus of mature IDUA is fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (mAb) against the murine transferrin receptor (TfR), and this fusion protein is designated cTfRMAb-IDUA. The cTfRMAb part of the fusion protein acts as a molecular Trojan horse to ferry the fused IDUA across the BBB and neuronal cell membrane via transport on the TfR. The IDUA enzyme activity of the fusion protein, 776 ± 79 units/µg protein, is comparable to recombinant IDUA. MPSI null mice, 6-8 months of age, were treated iv twice a week for 8 weeks with either saline or 1 mg/kg cTfRMAb-IDUA. The glycosoaminoglycan levels in liver, spleen, heart, and kidney were reduced by >95%, 80%, 36%, and 20%, respectively. Lysosomal inclusion bodies in the brain were quantitated from semithin sections stained with o-toluidine blue and normalized per 100 nucleoli per brain section. Treatment of the MPSI mice with the cTfRMAb-IDUA reduced intracellular lysosomal inclusion bodies by 73% in brain, as compared to the MPSI mice treated with saline. In conclusion, the reversal of pre-existing neural pathology in the brain of MPSI mice is possible with receptor-mediated enzyme replacement therapy of the brain.

[Relieving pre-exam anxiety syndrome with wrist-ankle acupuncture: a randomized controlled trial].

BACKGROUND: Pre-exam anxiety syndrome is a common condition occurring in pre-exam students and directly affects their examination performance and physical state. Wrist-ankle acupuncture has significant therapeutic effects in treating mental disorders and may also relieve the symptoms of pre-exam anxiety syndrome. OBJECTIVE: To assess the therapeutic effect of wrist-ankle acupuncture on pre-exam anxiety syndrome. DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: A total of 60 students who met the inclusion criteria of pre-exam anxiety syndrome were enrolled from a university in Shanghai and they were randomly divided into treatment group and control group. There were 30 cases in each group, and no case failed to follow-up. In the treatment group, wrist-ankle acupuncture was adopted to point upper 1 bilaterally (impression between flexor carpi ulnaris tendon and ulnar margin), and there was no requirement for Deqi (arrival of qi). In the control group, sham acupuncture was adopted. The treatment was applied 3 times totally in both groups one week before the exam, once every other day, each time with the needles retained for 30 min. MAIN OUTCOME MEASURES: The therapeutic effects were compared between two groups. Before and after 3 treatments, Sarason Test Anxiety Scale (TAS) and Expectation and Treatment Credibility Scale (ETCS) were measured and evaluated. RESULTS: The therapeutic effect experienced by the treatment group was better than that of the control group (P<0.05). There were no statistically significant differences in TAS and ETCS before treatment between the two groups. The scores of TAS after treatment in two groups were higher than those before treatment (P<0.05, P<0.01). There were statistical differences in TAS absolute difference and TAS relative difference between the two groups and the treatment group had better results (P<0.05, P<0.01). After treatment, patients in the treatment group had higher scores in ETCS than those in the control group (P<0.05, P<0.01). No adverse reaction was reported. CONCLUSION: Wrist-ankle acupuncture can relieve the symptoms of pre-exam anxiety syndrome significantly, and this therapy is highly safe.

Impacts of stand density on diversity of understory plant and soil seed banks in a Pinus massoniana plantation.

Stand density is a critical factor impacting the diversity of understory plants. We analyzed the diversity of understory plants and soil seed banks, as well as their relationship by setting up three planting densities in a Pinus massoniana plantation, including low density (1575 trees·hm-2, D1), medium (2474 trees·hm-2, D2), and high (3550 trees·hm-2, D3). It aimed to provide a scientific basis for the implementation of the multi-objective sustainable development of plantations. The results showed that there were 70 species of herbs and shrubs belonging to 42 families and 62 genera. D1 was dominated by heliophiles, whereas both the D2 and D3 were dominated by shade-tolerant species. The Margalef (M), Shannon (H), Simpson (D), Pielou (Jsw), and Altalo (Al) indices of the herbs and shrubs exhibited a downward trend with increasing stand den-sity. In the herb layer, D1 and D3 showed significant difference in H, D, Jsw and Al. There were significant differences of Jsw and Al in the shrub layer among the three stand densities, but no diffe-rence of H and D. H, D, Jsw and Al in the soil seed bank first decreased and then increased with increasing stand density, with species richness and diversity being the highest in D1. The similarity coefficient of Jaccard and Sorensen among different stand densities was low. In the herb layer, M was positively correlated with Jsw. The correlations between stand density and H, D, Jsw and Al were greater in the shrub layer than in the herb layer. There was significant negative correlation between stand density and Jsw both in the shrub and herb layers. The stand density of 1575 trees·hm-2 was comparatively beneficial for the development of understory, plant diversity, and sustainability of P. massoniana plantation.

Monoclonal antibody-glial-derived neurotrophic factor fusion protein penetrates the blood-brain barrier in the mouse.

Glial-derived neurotrophic factor (GDNF) is a potent neuroprotective agent for multiple brain disorders, including Parkinson's disease. However, GDNF drug development is difficult because GDNF does not cross the blood-brain barrier (BBB). To enable future drug development of GDNF in mouse models, the neurotrophin was re-engineered as an IgG fusion protein to enable penetration through the BBB after intravenous administration. The 134-amino acid GDNF was fused to the heavy chain of a chimeric monoclonal antibody (MAb) against the mouse transferrin receptor (TfR) designated the cTfRMAb. This antibody undergoes receptor-mediated transport across the BBB and acts as a molecular Trojan horse to ferry the GDNF into mouse brain. The cTfRMAb-GDNF fusion protein was expressed by stably transfected Chinese hamster ovary cells, affinity-purified, and the biochemical identity was confirmed by mouse IgG and GDNF Western blotting. The cTfRMAb-GDNF fusion protein was bifunctional and bound with high affinity to both the GDNF receptor alpha1, ED(50) = 1.7 +/- 0.2 nM, and the mouse TfR, ED(50) = 3.2 +/- 0.3 nM. The cTfRMAb-GDNF fusion protein was rapidly taken up by brain, and the brain uptake was 3.1 +/- 0.2% injected dose/g brain at 60 min after intravenous injection of a 1-mg/kg dose of the fusion protein. Brain capillary depletion analysis showed the majority of the fusion protein was transcytosed across the BBB with penetration into brain parenchyma. The brain uptake results indicate it is possible to achieve therapeutic elevations of GDNF in mouse brain with intravenous administration of the cTfRMAb-GDNF fusion protein.

Re-engineering erythropoietin as an IgG fusion protein that penetrates the blood-brain barrier in the mouse.

Erythropoietin (EPO) is a potent neuroprotective agent that could be developed for the treatment of multiple brain disorders. However, EPO does not cross the blood-brain barrier (BBB). A brain-penetrating form of EPO, specific for the mouse, was engineered by fusion of the 166 amino acid EPO to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), and this new fusion protein is designated cTfRMAb-EPO. The fusion protein was expressed in stably transfected Chinese hamster ovary cells and purified by protein G chromatography. The fusion protein was homogeneous on SDS-PAGE and Western blotting, and bound the mouse EPO receptor (EPOR) with high affinity, ED50 = 0.33 ± 0.04 nM. The cTfRMAb-EPO fusion protein was radiolabeled by tritiation and injected intravenously (iv) into adult mice for measurements of the plasma pharmacokinetics and brain uptake. The (3)H-fusion protein was rapidly cleared from the blood with a clearance rate of 5.9 ± 0.3 mL/min/kg, which is 14-fold faster than the clearance of EPO in the mouse. The cTfRMAb-EPO fusion protein penetrated the BBB in vivo, as shown by the capillary depletion method. The brain uptake of the cTfRMAb-EPO fusion protein was 2.0 ± 0.1% injected dose/g of brain following iv administration. The high level of brain uptake of the fusion protein enables pharmacologic increases in exogenous EPO in the mouse brain following the systemic injection of the cTfRMAb-EPO fusion protein. In conclusion, EPO has been re-engineered as an IgG fusion protein that binds dual receptors: the mouse TfR, to enable penetration of the BBB, and the mouse EPOR, to produce neuroprotection in brain behind the intact BBB.

Pharmacokinetics and brain uptake of a genetically engineered bifunctional fusion antibody targeting the mouse transferrin receptor.

Monoclonal antibodies (MAbs) are potential new therapeutics for brain diseases. However, MAbs do not cross the blood-brain barrier (BBB). The present work describes the genetic engineering of a fusion protein composed of a therapeutic single chain Fv (ScFv) antibody and a mouse/rat chimeric MAb against the mouse transferrin receptor (TfR). The TfRMAb acts as a molecular Trojan horse to ferry the therapeutic ScFv across the BBB in vivo in the mouse. The ScFv is fused to the carboxyl terminus of the heavy chain of the chimeric TfRMAb, and this fusion protein is designated cTfRMAb-ScFv. Chinese hamster ovary cells were permanently transfected, and a high secreting cell line in serum free medium was cloned. The cTfRMAb-ScFv fusion protein was purified to homogeneity on gels and Western blotting with protein G affinity chromatography. The cTfRMAb-ScFv fusion protein was bifunctional and bound both the target antigen, as determined by ELISA, and the mouse TfR, as determined with a radio-receptor assay. The cTfRMAb-ScFv fusion protein was radio-iodinated with the Bolton-Hunter reagent, and a pharmacokinetics study in mice showed that the fusion protein was rapidly cleared from blood with a median residence time of 175 +/- 32 min. The fusion protein was avidly taken up by brain with a % injected dose (ID)/g of 3.5 +/- 0.7, as compared to an MAb with no receptor specificity, which was 0.06 +/- 0.01% ID/g. These studies demonstrate that therapeutic MAbs may be re-engineered as fusion proteins with BBB molecular Trojan horses for targeted delivery across the BBB in vivo.