A new metal ion chelator attenuates human tau accumulation-induced neurodegeneration and memory deficits in mice.

Neuronal neurofibrillary tangles containing Tau hyperphosphorylation proteins are a typical pathological marker of Alzheimer's disease (AD). The level of tangles in neurons correlates positively with severe dementia. However, how Tau induces cognitive dysfunction is still unknown, which leads to a lack of effective treatments for AD. Metal ions deposition occurs with tangles in AD brain autopsy. Reduced metal ion can improve the pathology of AD. To explore whether abnormally phosphorylated Tau causes metal ion deposition, we overexpressed human full-length Tau (hTau) in the hippocampal CA3 area of mice and primary cultured hippocampal neurons (CPHN) and found that Tau accumulation induced iron deposition and activated calcineurin (CaN), which dephosphorylates glycogen synthase kinase 3 beta (GSK3ß), mediating Tau hyperphosphorylation. Simultaneous activation of CaN dephosphorylates cyclic-AMP response binding protein (CREB), leading to synaptic deficits and memory impairment, as shown in our previous study; this seems to be a vicious cycle exacerbating tauopathy. In the current study, we developed a new metal ion chelator that displayed a significant inhibitory effect on Tau phosphorylation and memory impairment by chelating iron ions in vivo and in vitro. These findings provide new insight into the mechanism of memory impairment induced by Tau accumulation and develop a novel potential treatment for tauopathy in AD.

Steriod for Autoimmune pancreatitis complicating by gastric varices: A case report.

BACKGROUND: Both autoimmune pancreatitis (AIP) and gastric varices are related to various diseases. However, AIP complicated by gastric varices is extremely rare, and has only been reported in a few studies. Here, we report a case of AIP complicated by gastric varices in a female Chinese patient. CASE SUMMARY: A 59-year-old Chinese woman was admitted to our hospital with mild abdominal pain. Computed tomography and magnetic resonance cholangiopancreatography revealed a diffusely enlarged pancreas, an obstructed splenic vein and slight splenomegaly. Esophagogastroduodenoscopy showed gastric varices in the partial gastric fundus and the red-color sign was positive. Blood chemistry showed that IgG4 was notablely elevated. The patient was diagnosed with AIP complicated by gastric varices. Steroid therapy was administered to this patient with the risk of gastrointestinal bleeding. After one year of therapy, the pancreas, spleen and splenic vein recovered to the normal states, and the gastric varices had disappeared. CONCLUSION: We present this case together with evidence from the literature to demonstrate the effectiveness of steroid therapy in the treatment of AIP complicated by gastric varices.

[Effect of electroacupuncture on complement C1q and microglia phagocytosis in hippocampus of SAMP8 mice].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression of Iba-1, complement C1q and CD68 in hippocampus of SAMP8 mice, so as to explore its mechanisms underlying improvement of Alzheimer's disease (AD). METHODS: Twenty-four male SAMP8 mice were randomly and equally divided into model and EA groups, and 12 SAMR1 mice were used as the control group. EA (2 Hz, 1.5-2.0 mA) was applied to "Baihui" (GV20), "Dazhui"(GV14) and "Shen-shu"(BL23) for 20 min once daily in the EA group, each course of treatment was 8 days, with an interval of 2 days between two courses, and the mice were treated for 3 courses. Morris water maze test was performed to assess the learning-memory ability of mice. The positive expression levels of Iba-1 and CD68 proteins in the hippocampus CA1 region were detected by immunohistochemistry. The mRNA and protein expression levels of Iba-1,C1q and CD68 in the hippocampus were detected by real-time PCR and Western blot, separately. RESULTS: Compared with the control group, the average escape latency of Morris water maze test was prolonged in the model group (P<0.01), duration of swimming in the original platform quadrant and the number of original platform crossing were significantly shorter and decreased respectively (P<0.01). Compared with the model group, the average escape latency in the EA group was shortened (P<0.05, P<0.01), the duration of swimming in the original platform quadrant and the number of original platform crossing were significantly prolonged and increased (P<0.01). The immunoactivity of Iba-1 and CD68 in hippocampal CA1 region, and mRNA and protein expression levels of hippocampal Iba-1,C1q and CD68 were significantly up-regulated in the model group in contrast to the control group (P<0.01, P<0.05), and obviously down-regulated except the mRNA expression level of hippocampal Iba-1 in the EA group relevant to the model group (P<0.01, P<0.05). CONCLUSION: EA can improve the learning and memory ability of SAMP8 mice, which may be associated with its effect in inhibiting of complement C1q-dependent microglial phagocytosis in the hippocampus.

Gastroprotective and anti-ulcer effects of oxymatrine against several gastric ulcer models in rats: Possible roles of antioxidant, antiinflammatory, and prosurvival mechanisms.

Oxymatrine (OXY) has antioxidative and antiinflammatory activities. In the present work, we investigate the effects of OXY on gastric ulcer models and elucidate the underlying mechanisms of action. Ethanol, indometacin, and restraint water immersion stress-induced ulcerated models were used. The ulcer area was measured, and samples of gastric tissue were taken for pathological, histochemical, and biochemical analyses. OXY effectively reduced the area of gastric ulcers and improved the pathological changes of ulcerated tissue. OXY enhanced expression of Bcl-2, reduced Bax protein expression, and inhibited alcohol-induced apoptotic death in both ulcerated tissue and human gastric epithelial cells. OXY increased the prostaglandin E2 level and improved oxidative stress (malondialdehyde, superoxide dismutase, catalase, and nitric oxide) and inflammatory parameters (TNF-a, IL-6, and IL-1) of ulcer tissue. OXY prevented an inflammatory response via decreasing expression of p38, p-ERK, p-JNK, and inhibiting NF-κB p65 translocation from the cytoplasm to the nucleus. Our results reveal that OXY has remarkable protective effects on gastric ulcers. The action of OXY may be mediated via suppression of gastric inflammatory reactions, oxidative stress, and pro-apoptotic actions, which were the results of blockades of MAPKs and NF-κB signaling pathways. Our results provide evidence for the beneficial effects of OXY for treating peptic ulcers.

ICAM-1 Deficiency in the Bone Marrow Niche Impairs Quiescence and Repopulation of Hematopoietic Stem Cells.

The bone marrow niche plays a critical role in controlling the fate of hematopoietic stem cells (HSCs) by integrating intrinsic and extrinsic signals. However, the molecular events in the HSC niche remain to be investigated. Here, we report that intercellular adhesion molecule-1 (ICAM-1) maintains HSC quiescence and repopulation capacity in the niche. ICAM-1-deficient mice (ICAM-1-/-) displayed significant expansion of phenotypic long-term HSCs with impaired quiescence, as well as favoring myeloid cell expansion. ICAM-1-deficient HSCs presented normal reconstitution capacity during serial transplantation; however, reciprocal transplantation experiments showed that ICAM-1 deficiency in the niche impaired HSC quiescence and repopulation capacity. In addition, ICAM-1 deletion caused failure to retain HSCs in the bone marrow and changed the expression profile of stroma cell-derived factors, possibly representing the mechanism for defective HSCs in ICAM-1-/- mice. Collectively, these observations identify ICAM-1 as a regulator in the bone marrow niche.

Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells.

Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human ß-interferon and ß-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

[Enhanced nutritional therapy may promote wound healing after endoscopic therapy in patients with liver cirrhosis and esophageal varices].

OBJECTIVE: To investigate the effect of enhanced nutritional therapy on wound healing after endoscopic therapy in patients with liver cirrhosis and esophageal varices. METHODS: Fifty patients with liver cirrhosis and esophageal varices were randomly divided into an enhanced nutritional therapy group (n = 25) and a control group (n = 25). The enhanced nutritional therapy group received one week of enhanced nutritional supplementation, including liver nutritional elements, prior to routine endoscopic therapy. The routine without any change to their diet. The rate of transformation and status of wound healing of esophageal varices were compared between the two groups. RESULTS: The ratio of ulcers occurring at the injection site was lower in the enhanced nutrition group than in the control group (16/25 vs. 23/25; x2 = 5.711, P = 0.017). The enhanced nutrition group had only one case of minimal bleeding occurring during endoscopy as compared to the seven cases of bleeding in the control group (x2 = 5.357, P = 0.021). On average, the enhanced nutrition group required less sessions of endoscopic treatment to achieve eradication of esophageal varices than the control group (3.8 vs. 4.1; t = 2.069, P = 0.044). CONCLUSION: Pre-endoscopic enhanced nutritional therapy may benefit patients with liver cirrhosis and esophageal varices by promoting recovery of procedure-related local tissue injury and occlusion of varices.

Antiviral therapy delays esophageal variceal bleeding in hepatitis B virus-related cirrhosis.

AIM: To investigate the effect of antiviral therapy with nucleoside analogs in hepatitis B virus (HBV)-related cirrhosis and esophageal varices. METHODS: Eligible patients with HBV-related cirrhosis and esophageal varices who consulted two tertiary hospitals in Beijing, China, the Chinese Second Artillery General Hospital and Chinese PLA General Hospital, were enrolled in the study from January 2005 to December 2009. Of 117 patients, 79 received treatment with different nucleoside analogs and 38 served as controls. Bleeding rate, change in variceal grade and non-bleeding duration were analyzed. Multivariate Cox proportional hazard regression was used to identify factors related to esophageal variceal bleeding. RESULTS: The bleeding rate was decreased in the antiviral group compared to the control group (29.1% vs 65.8%, P < 0.001). Antiviral therapy was an independent factor related to esophageal bleeding in multivariate analysis (HR = 11.3, P < 0.001). The mean increase in variceal grade per year was lower in the antiviral group (1.0 ± 1.3 vs 1.7 ± 1.2, P = 0.003). Non-bleeding duration in the antiviral group was prolonged in the Kaplan-Meier model. Viral load rebound was observed in 3 cases in the lamivudine group and in 1 case in the adefovir group, all of whom experienced bleeding. Entecavir and adefovir resulted in lower bleeding rates (17.2% and 28.6%, respectively) than the control (P < 0.001 and P = 0.006, respectively), whereas lamivudine (53.3%) did not (P = 0.531). CONCLUSION: Antiviral therapy delays the progression of esophageal varices and reduces bleeding risk in HBV-related cirrhosis, however, high-resistance agents tend to be ineffective for long-term treatment.

Simultaneous expression of displayed and secreted antibodies for antibody screen.

The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS) analysis, as well as in conditioned medium in a secreted version for function analysis.

Saikosaponin a Enhances Transient Inactivating Potassium Current in Rat Hippocampal CA1 Neurons.

Saikosaponin a (SSa), a main constituent of the Chinese herb Bupleurum chinense DC., has been demonstrated to have antiepileptic activity. Recent studies have shown that SSa could inhibit NMDA receptor current and persistent sodium current. However, the effects of SSa on potassium (K(+)) currents remain unclear. In this study, we tested the effect of SSa on 4AP-induced epileptiform discharges and K(+) currents in CA1 neurons of rat hippocampal slices. We found that SSa significantly inhibited epileptiform discharges frequency and duration in hippocampal CA1 neurons in the 4AP seizure model in a dose-dependent manner with an IC 50 of 0.7 µ M. SSa effectively increased the amplitude of I Total and I A , significantly negative-shifted the activation curve, and positive-shifted steady-state curve of I A . However, SSa induced no significant changes in the amplitude and activation curve of I K . In addition, SSa significantly increased the amplitude of 4AP-sensitive K(+) current, while there was no significant change in the amplitude of TEA-sensitive K(+) current. Together, our data indicate that SSa inhibits epileptiform discharges induced by 4AP in a dose-dependent manner and that SSa exerts selectively enhancing effects on I A . These increases in I A may contribute to the anticonvulsant mechanisms of SSa.

Association of chemotherapy-induced leucopenia with treatment outcomes in advanced non-small-cell lung cancer cases receiving the NP regimen.

BACKGROUND: Chemotherapy induced leutropenia has been shown to be associated with improved treatment outcomes in selected solid tumors. We studied the association of chemotherapy induced leutropenia with treatment related outcomes in advanced non-small-cell lung cancer. METHODS: This is a prospective analysis of patients receiving chemotherapy for advanced NSCLC at the Shandong Cancer Hospital from 2005-07.The chemotherapy included cisplatin 35 mg/m2, IV on d1,2 and vinorelbine 25 mg/m2 IV on d1,8 every 21 days. Patients were stratified into three groups (A) those experiencing grades 0 leucopenia, group (B) grades 1-2 and group (C) grades 3-4. The outcomes studied were response rate (RR), disease control rate (DCR), and time to progression (TTP). RESULTS: 128 patients were studied. The RRs in groups A, B and C were 30.8%, 56.8% and 71.4%, respectively, p=0.010. The DCRs were 61.5%, 83.8% and 92.9%, respectively, p=0.009 and the median TTPs were 150 days (95%CI: 91-209), 189 days (95%CI: 181-197) and 207 days (95%CI: 172-242), p=0.009. The differences in RR and TTP were significant. In patients whose CIL kept on 10 days at least, the TTP was significantly prolonged, p=0.0213, and the same was the case for those experiencing grades 1-2 leucopenia and ECOG 0, p=0.0412. CONCLUSIONS: Occurrence of CIL correlated with RR and TTP in patients with advanced NSCLC receiving cisplatin and vinorelbine chemotherapy, especially in patients experiencing grades 1-2 leucopenia and ECOG 0, and the same for those with CIL persisting for 10 days at least. CIL could be a biological measure of drug activity and a marker of efficacy.

Identification of HBsAg-specific antibodies from a mammalian cell displayed full-length human antibody library of healthy immunized donor.

Hepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified. Combining four-way ligation and the Flp recombinase-mediated integration (Flp-In) system, we constructed a mammalian cell-based, fully human, full-length antibody display library in which each cell displayed only one kind of antibody molecule. By screening the cell library using fluorescence-activated cell sorting (FACS), eight cell clones that displayed HBsAg-specific antibodies on cell surfaces were identified. DNA sequence analysis of the antibody genes revealed three unique antibodies. FACS data indicated that fluorescent strength of expression (FSE), fluorescent strength of binding (FSB) and relative binding ability (RBA) were all different among them. These results demonstrated that by using our antibody mammalian display and screening platform, we can successfully identify antigen-specific antibodies from an immunized full-length antibody library. Therefore, this platform is very useful for the development of therapeutic antibodies.

[Construction of rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries].

OBJECTIVE: To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries. METHODS: Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR. The amplified LCκ and VH genes were inserted into the vector pDGB-HC-TM separately, and the ligated libraries were transformed into competent E.coli TOPO-10 strain to construct the rheumatoid arthritis-specific antibody heavy and light chain libraries. 293T cells were co-transfected with the libraries and the full-length fully human antibody expressed on the surface of 293T cells were analyzed by flow cytometry. RESULTS: The libraries of rheumatoid arthritis-specific antibody LCκ and heavy chain (IgG1) were constructed. The expression of full-length fully human antibody on the surface of 293T cells was confirmed by flow cytometry. With the rates of correct LCκ and heavy chain sequence insertion reaching 80% and 60%, respectively, as shown by DNA sequence analysis of the randomly selected clones, the libraries showed an expressible combinatory diversity of 6.13×10(10). CONCLUSION: The constructed libraries provide a useful platform for screening rheumatoid arthritis-specific antibodies.

Diagnosis and management of acute variceal bleeding: Asian Pacific Association for Study of the Liver recommendations.

BACKGROUND: Acute variceal bleeding (AVB) is a medical emergency and associated with a mortality of 20% at 6 weeks. Significant advances have occurred in the recent past and hence there is a need to update the existing consensus guidelines. There is also a need to include the literature from the Eastern and Asian countries where majority of patients with portal hypertension (PHT) live. METHODS: The expert working party, predominantly from the Asia-Pacific region, reviewed the existing literature and deliberated to develop consensus guidelines. The working party adopted the Oxford system for developing an evidence-based approach. Only those statements that were unanimously approved by the experts were accepted. RESULTS: AVB is defined as a bleed in a known or suspected case of PHT, with the presence of hematemesis within 24 h of presentation, and/or ongoing melena, with last melanic stool within last 24 h. The time frame for the AVB episode is 48 h. AVB is further classified as active or inactive at the time of endoscopy. Combination therapy with vasoactive drugs (<30 min of hospitalization) and endoscopic variceal ligation (door to scope time <6 h) is accepted as first-line therapy. Rebleeding (48 h of T (0)) is further sub-classified as very early rebleeding (48 to 120 h from T (0)), early rebleeding (6 to 42 days from T (0)) and late rebleeding (after 42 days from T (0)) to maintain uniformity in clinical trials. Emphasis should be to evaluate the role of adjusted blood requirement index (ABRI), assessment of associated comorbid conditions and poor predictors of non-response to combination therapy, and proposed APASL (Asian Pacific Association for Study of the Liver) Severity Score in assessing these patients. Role of hepatic venous pressure gradient in AVB is considered useful. Antibiotic (cephalosporins) prophylaxis is recommended and search for acute ischemic hepatic injury should be done. New guidelines have been developed for management of variceal bleed in patients with non-cirrhotic PHT and variceal bleed in pediatric patients. CONCLUSION: Management of acute variceal bleeding in Asia-Pacific region needs special attention for uniformity of treatment and future clinical trials.

[Rapid construction of a mammalian cell surface display library of full-length human antibodies].

OBJECTIVE: To construct a mammalian cell surface display library of full-length human antibodies. METHODS: The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Cκ) of the antibodies were amplified by RT-PCR. The amplified VH and Cκ gene sequences were separately inserted into the vector pDGB-HC-TM. The ligation mixtures were transformed into competent E.coli DH5α cells to construct the antibody libraries, and the library sizes and diversity were analyzed. The library DNAs were transfected into CHO cells and the expression of the full-length human antibodies on the surface of CHO cells was analyzed by flow cytometry. RESULTS: The heavy chain gene library constructed showed a diversity of 2.6 × 10(5), and the kappa light chain gene library had a diversity of 2.0 × 10(5). Sequence analysis of 10 clones randomly selected from the constructed heavy chain gene library and 10 from the light chain gene library showed that 8 heavy chain clones and all 10 light chain clones contained correct open reading frames. Flow cytometry demonstrated that all the 18 clones expressed full-length antibodies, which could be detected on CHO cell surfaces. After co-transfection of the heavy chain and light chain gene libraries into CHO cells, the expression of full-length antibodies on CHO cell surfaces was detected with the positive cells reaching 31.5. CONCLUSIONS: A full-length human mammalian display antibody library with a combinatory diversity of 5.2 × 10(10) can be constructed in two weeks, which allows the display of full-length antibodies on mammalian cell surface.

[Bioinformatic analysis of genes related to temporal epilepsy].

OBJECTIVE: To investigate the genes associated with temporal epilepsy and explore the molecular mechanism of epilepsy. METHODS: The microarray data of temporal epilepsy were downloaded from the Gene Expression Omnibus (GEO) database and analyzed by bioinformatics methods using String, KEGG and Panther databases. RESULTS: Of all the 71 differentially expressed genes, 51 were found to encode proteins with interactions; the main biological pathways involved included neuroactive ligand-receptor interactions, MAPK signaling pathway, and calcium signaling pathway etc. CONCLUSION: The pathogenesis of epilepsy involves multiple genes, and investigations of these genes may provide valuable insights into the mechanism of epilepsy.

Four-way ligation for construction of a mammalian cell-based full-length antibody display library.

A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces. Using this strategy, only 2 weeks were required to successfully construct high-quality, full-length human antibody libraries.

A novel strategy for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfaces.

Development of a versatile mammalian display system is essential for the selection of functional human antibodies with high affinities. Here we described a novel strategy for rapid construction of full-length antibody libraries that could be efficiently expressed on mammalian cell surfaces. The universal vector pDGB-HC-TM was constructed by inserting multiple cloning site unique sequences recognized by restriction endonucleases BsmBI, SfiI, and BstXI for the pop-in and pop-out of genes of interest. Cytomegalovirus promoter, a commonly used promoter for high expression of proteins in a variety of mammalian cells, was used to drive expression of the inserted antibody genes and a transmembrane domain from platelet-derived growth factor receptor was fused in frame to the C-terminus of heavy chain consistent region to anchor the antibody expressed on the mammalian cell surface. Using this strategy, we constructed a full-length human antibody display library. DNA sequence analysis and expression analysis indicated that the library constructed had a combinatory expressible, detectable diversity of 6.58 x 10(10).