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Background: Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health., Methodology/principal Findings: To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms., Conclusions/significance: These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. PD-1 and its ligands are broadly expressed and exert a wider range of immunoregulatory roles in T cells activation and tolerance compared with other CD28 members. Subsequent studies show that PD-1-PD-L interaction regulates the induction and maintenance of peripheral tolerance and protect tissues from autoimmune attack. PD-1 and its ligands are also involved in attenuating infectious immunity and tumor immunity, and facilitating chronic infection and tumor progression. The biological significance of PD-1 and its ligand suggests the therapeutic potential of manipulation of PD-1 pathway against various human diseases. In this review, we summarize our current understanding of PD-1 and its ligands ranging from discovery to clinical significance.

Experimental pancreatitis is associated with activation of polyamine catabolism. The polyamine analog bismethylspermine (Me(2)Spm) can ameliorate pancreatic injury. We investigated the roles of polyamine catabolism in remote organs during pancreatitis and explored the mechanism of polyamine catabolism by administering Me(2)Spm. Acute pancreatitis was induced by an infusion of 2 or 6% taurodeoxycholate before Me(2)Spm administration. Blood, urine and tissues were sampled at 24 and 72 h to assess multi-organ injury and polyamine catabolism. The effect of Me(2)Spm on mortality in experimental pancreatitis was tested separately. Liver putrescine levels were elevated following liver injury. Me(2)Spm increased the activity of spermidine/spermine N(1)-acetyltransferase (SSAT) and depleted the spermidine, spermine or putrescine levels. Lung putrescine levels increased, and SSAT and spermine decreased following lung injury. Me(2)Spm enhanced the activity of SSAT and decreased the spermidine and spermine levels. Renal injury was manifested as an increase in creatinine or a decrease in urine output. Decreases in kidney SSAT, spermidine or spermine and an increase in putrescine were found during pancreatitis. In the 2% taurodeoxycholate model, Me(2)Spm decreased urine output and raised plasma creatinine levels. Me(2)Spm increased SSAT and decreased polyamines. Excessive Me(2)Spm accumulated in the kidney, and greater amounts were found in the 6% taurodeoxycholate model in which this mortality was not reduced by Me(2)Spm. In the 2% taurodeoxycholate model, Me(2)Spm dose-dependently induced mortality at 72 h. Like pancreatic injury, remote organ injury in pancreatitis is associated with increased putrescine levels. However, Me(2)Spm could not ameliorate multi-organ injury. Me(2)Spm administration was associated with significant renal toxicity and induced mortality, suggesting that the current dose is too high and needs to be modified.

Background: Overinduced polyamine catabolism (PC) in a transgenic rat model has been suggested to be a mediator of trypsin activation which is important in acinar cell necrosis. PC has also been observed in experimental taurodeoxycholate pancreatitis. We hypothesized that PC may be a mediator of trypsin activation in taurodeoxycholate pancreatitis., Methods: Pancreatitis was induced in wild-type rats by 2 or 6% taurodeoxycholate infusion or in transgenic rats by overexpressing spermidine/spermine N(1)-acetyltransferase (SSAT). The time courses of necrosis, caspase-3 immunostaining, SSAT, polyamine levels, and trypsinogen activation peptide (TAP) were monitored. The effect of the polyamine analogue bismethylspermine (Me(2)Spm) was investigated., Results: In a transgenic pancreatitis model, TAP and acinar necrosis increased simultaneously after the activation of SSAT, depletion of spermidine, and development of apoptosis. In taurodeoxycholate pancreatitis, necrosis developed along with the accumulation of TAP. SSAT was activated simultaneously or after TAP accumulation and less than in the transgenic model, with less depletion of spermidine than in the transgenic model. Supplementation with Me(2)Spm ameliorated the extent of acinar necrosis at 24 h, but contrary to previous findings in the transgenic model, in the taurodeoxycholate model it did not affect trypsin activation. Compared with the transgenic model, no extensive apoptosis was found in taurodeoxycholate pancreatitis., Conclusions: Contrary to transgenic SSAT-overinduced pancreatitis, PC may not be a mediator of trypsin activation in taurodeoxycholate pancreatitis. The beneficial effect of polyamine supplementation on necrosis in taurodeoxycholate pancreatitis may rather be mediated by other mechanisms than amelioration of trypsin activation. and IAP., (Copyright © 2011 S. Karger AG, Basel.)

Inhibitory receptors play a crucial role in regulating CD8 T-cell function during chronic viral infection. T-cell Ig- and mucin-domain-containing molecule-3 (Tim-3) is well known to negatively regulate T-cell responses, but its role in CD8 T-cell exhaustion during chronic infection in vivo remains unclear. In this study, we document coregulation of CD8 T cell exhaustion by Tim-3 and PD-1 during chronic lymphocytic choriomeningitis virus infection. Whereas Tim-3 was only transiently expressed by CD8 T cells after acute infection, virus-specific CD8 T cells retained high Tim-3 expression throughout chronic infection. The majority (approximately 65% to 80%) of lymphocytic choriomeningitis virus-specific CD8 T cells in lymphoid and nonlymphoid organs coexpressed Tim-3 and PD-1. This coexpression of Tim-3 and PD-1 was associated with more severe CD8 T-cell exhaustion in terms of proliferation and secretion of effector cytokines such as IFN-gamma, TNF-alpha, and IL-2. Interestingly, CD8 T cells expressing both inhibitory receptors also produced the suppressive cytokine IL-10. Most importantly, combined blockade of Tim-3 and PD-1 pathways in vivo synergistically improved CD8 T cell responses and viral control in chronically infected mice. Taken together, our study defines a parameter for determining the severity of CD8 T cell dysfunction and for identifying virus-specific CD8 T cells that produce IL-10, and shows that targeting both PD-1 and Tim-3 is an effective immune strategy for treating chronic viral infections.

As a pre-digestion method of breast milk microwave-digestion was adopted in this method, and the contents of calcium and phosphorus were determined by ICP-MS at the same time. By optimizing conditions of digestion and ICP-MS, appropriate internal elements and the isotope mass number and EPA200.8 interference calibration equation were chosen. The accuracy and reliability of method were verified through the recovery and milk powder analysis of national standard substances (GBW10017). Determination results are in the permissible range. The relative deviation (RSD) is less than 2.40%, while the recovery is between 102.8% and 104.0%. ICP-MS method has wide dynamic range, and doesn't need to dilute samples. It is suitable for lots of samples and multi-element analyzed at the same time, and making up default of different method and different element determined respectively in national standard. It is a determination method for calcium and phosphorus supply of breast milk.

Cell-permeable peptides (CPPs) promote the transduction of nonpermissive cells by recombinant adenovirus (rAd) to improve the therapeutic efficacy of rAd. In this study, branched oligomerization of CPPs significantly enhanced the transduction of human mesenchymal stem cells (MSCs) by rAd in a CPP type-independent manner. In particular, tetrameric CPPs increased transduction efficiency at 3000-5000-fold lower concentrations than did monomeric CPPs. Although branched oligomerization of CPPs also increases cytotoxicity, optimal concentrations of tetrameric CPPs required for maximum transduction are at least 300-1000-fold lower than those causing 50% cytotoxicity. Furthermore, although only approximately 60% of MSCs were maximally transduced at 500 muM of monomeric CPPs, >95% of MSCs were transduced with 0.1 muM of tetrameric CPPs. Tetrameric CPPs also significantly increased the formation and net surface charge of CPP/rAd complexes, as well as the binding of rAd to cell membranes at a greater degree than did monomeric CPPs, followed by rapid internalization into MSCs. In a critical-size calvarial defect model, the inclusion of tetrameric CPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs that were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tetrameric CPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tetrameric CPPs to provide an innovative tool for MSC-based gene therapy and for in vitro gene delivery to MSCs.

A simple and reproducible method was developed for the quantification of ketamine and S(+)-ketamine in dog plasma using a high-performance liquid chromatography system coupled to a positive ion electrospray mass spectrometric analysis. Solid-phase extraction was used for extracting analytes from dog plasma samples. The analytes were separated on a Zorbax SB C(18) column (100 x 2.1 mm, 3.5 microm) with acetonitrile-formate buffer (10 mM ammonium formate and 0.3% formic acid) (17 : 83, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was operated under selected ion monitoring mode. [M + H](+) at m/z 238 for ketamine and S(+)-ketamine and [M + H](+) at m/z 180 for phenacetin (internal standard) were selected as detecting ions, respectively. The method was linear in the concentration range 51.6-2580 ng/mL. The intra- and inter-day precisions (RSD %) were within 11.3% and the assay accuracies ranged from 80.0 to 101.4%. Their average recoveries were greater than 91.1% at all test concentrations. The analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the toxicokinetics study and comparison of ketamine and S (+)-ketamine following intravenous administration to dogs., (2009 John Wiley & Sons, Ltd.)

IL-7 plays a crucial role in the homeostatic proliferation, differentiation and survival of T cells, as well as in the survival and proliferation of precursor B cells. Here, we demonstrated that utilizing nonlytic Fc-fused IL-7 (IL-7-Fc(m)) as a genetic adjuvant significantly enhanced not only CD4(+) but also CD8(+) T-cell responses by E7 DNA immunization, in addition to improving protection against TC-1-induced tumors in comparison to IL-7 alone. Similar results were obtained in OT-1 adoptive transfer experiments with OVA DNA injection, suggesting independence from antigenic nature and experimental conditions. In particular, the increased frequency of CD8(+) T cells was mainly due to enhanced T-cell proliferation in T-cell priming, and not to decreased cellular apoptosis. Interestingly, the enhanced adjuvant effect was not seen in the co-delivery of lytic Fc-fused IL-7 (IL-7-Fc) which increases T-cell apoptosis as well as T-cell proliferation, suggesting that the T-cell proliferative effect may be neutralized by T-cell apoptosis. Thus, our findings suggest that nonlytic Fc, in contrast to lytic Fc, fusion to cytokines may provide an insight in designing a potent genetic adjuvant for inducing CD4(+) and CD8(+) T-cell responses.

Objective: On the basis of YouGui drink ([Chinese characters: see text]) to act on the normal rats in vitro osteoblast proliferation and differentiation, further observe YooGui drink ([Chinese characters: see text]) inductive effect for steroid induced necrosis of femoral head rats in vitro on cultivation of osteoblast proliferation and differentiation., Methods: Prednisolone acetate 49 mg/kg x d were injected into gluteal of 20 male SD rats (body weight in 100-120 g), continuously 6 days for model. After 7 days, under sterile conditions from the model rats bone marrow stromal cells were induced and cultured to osteoblast, and then randomly divided into 4 groups, respectively, add the high, medium and low concentration YouGui drink ([Chinese characters: see text]) containing serum (group R1, R2, R3) and control serum (group R4). After 72 h, osteoblast proliferation, alkaline phosphatase activities (APA) were detected, and expression level of osteoprotegerin (OPG) and osteoclast differentiation factor (RANKL) were detected by real-time fluorescence quantitative PCR., Results: The high and medium concentration YouGui drink ([Chinese characters: see text]) containing serum group compared with the control group (R1, R2 vs. R4), the rate of osteoblast proliferation was significantly high (P < 0.01), APA significantly increased (P < 0.01), relative expression level of osteoblast OPG mRNA significantly increased (P < 0.01), and with the concentration of a certain dose was positively correlated; RANKL mRNA significantly was lower (P < 0.01). The low concentration YouGui drink (t 91 t) containing serum group compared with the control group (R3 vs R4), there was no significantly different in the rate of osteoblast proliferation, ALP and relative expression level of osteoblast OPG mRNA, RANKL mRNA., Conclusion: The high concentration of YouGui drink ([Chinese characters: see text]) containing serum can obviously promote the proliferation and differentiation of steroid induced necrosis of femoral head rats in vitro osteoblast, and plays a clear role in promoting and enhance the relative expression of osteoblast OPG mRNA and an inhibitory effect on RANKL mRNA. The effect of YouGui drink ([Chinese characters: see text]) containing serum acting on induced in vitro cultured osteoblasts have relevance with dose, at a certain concentration range it can promote osteoblast differentiation and proliferation.

Purpose: The authors sought to quantitatively analyse enhancement characteristics of pancreatic insulinomas in different phases and determine the value of multidetector-row computed tomography (CT) for detecting insulinomas., Materials and Methods: Forty-six patients with surgically proven insulinomas diagnosed between 2002 and 2007 were retrospectively reviewed. These patients underwent single-phase (group 1) or dual-phase (group 2) helical CT scanning., Results: Sensitivity for detecting insulinomas in group 2 was superior to that in group 1 (p<0.05).The sensitivity for insulinoma detection in the arterial phase was superior to that in the portal-venous phase (p<0.05). The mean attenuation values of the insulinomas and normal pancreas during the unenhanced arterial and portal-venous phases were, respectively, 40.5+/-8.75 HU (Hounsfield units), 114.48+/-27.30 HU, 112.19+/-19.52 HU and 44.56+/-6.48 HU, 81.16+/-15.22 HU, 90.54+/-13.80 HU, and there was statistical difference between them (p=0.000). The contrast enhancement of insulinomas in the arterial and portal-venous phases was 74.03+/-29.51 HU and 70.90+/-21.93 HU, respectively, and there was no statistical difference between them (p=0.499). The tumour to normal-pancreas attenuation differences in the arterial and portal-venous phases were respectively 33.32+/-20.96 HU and 20.58+/-16.32 HU, respectively, and there was statistical difference between them (p=0.011)., Conclusions: Dual-phase CT has a promising sensitivity in detecting pancreatic insulinomas. The acquisition of images in the arterial phase is more helpful for detecting insulinomas.

DNA immunization induces B and T cell responses to various pathogens and tumors. However, these responses are known to be relatively weak and often transient. Thus, novel strategies are necessary for enhancing immune responses induced by DNA immunization. Here, we demonstrated that co-immunization of influenza virus nucleoprotein (NP) gene significantly enhances humoral and cell-mediated responses to codelivered antigens in mice. We also found that NP DNA coimmunization augments in vivo proliferation of adoptively transferred antigen-specific CD4 and CD8 T cells, which enhanced protective immunity against tumor challenge. Our results suggest that NP DNA can serve as a novel genetic adjuvant in cocktail DNA vaccination.

Since human papillomavirus (HPV) E6 and E7 are promising tumor antigens, we engineered E6 and E7 antigens to generate an optimal HPV DNA vaccine by codon optimization (Co), fusion of E6 and E7, addition of a tissue plasminogen activator (tpa) signal sequence, addition of CD40 ligand (CD40L) or Fms-like tyrosine kinase-3 ligand (Flt3L). The resulting constructs were investigated in terms of their antitumor activity as well as induction of HPV-specific CD8(+) T cell responses. When E6(Co) and E7(Co) were fused (E67(Co)), CD8(+) T cell responses specific for E6 or E7 antigen decreased, but the preventive antitumor effect rather improved, demonstrating the importance of broad immunity. Interestingly, Flt3L-fused HPV DNA vaccine exhibited stronger E6- and E7-specific CD8(+) T cell responses as well as therapeutic antitumor effect than that of CD40L linked HPV DNA vaccine. Finally, the optimal construct, tFE67(Co), was generated by including tpa signal sequence, Flt3L, fusion of E6 and E7, and codon optimization, which induces 23 and 25 times stronger E6- and E7-specific CD8(+) T cell responses than those of initial E67 fusion construct. In particular, inclusion of electroporation in intramuscular immunization of tFE67(Co) further enhances HPV-specific CD8(+) T cell responses, leading to complete tumor regression in a therapeutic setting. Thus, our results provide valuable insight on effective HPV DNA vaccine design and suggest that tFE67(Co) delivered with electroporation may be a promising therapeutic HPV DNA vaccine against cervical cancer.

Objectives: The present study was performed to compare the pancreatic injury response on the parenchymal resection either with ultrasonic scissors, electrocautery, or surgical scalpel., Methods: A 1 x 0.5 cm piece of rat pancreas was resected from side of the pancreas either with ultrasonic scissors (Harmonic Scalpel; UltraCision, Ethicon Endosurgery Inc., Cincinnati, OH) or electrocautery (Force FX; Valleylab, Tyco Healthcare Group LP, Boulder, CO) at two power levels, 1 and 3; 8W and 25W, respectively, or with surgical scalpel. Hemostasis was provided after surgical scalpel either with cellulose patch (Interceed; Johnson and Johnson Medical, Inc., New Brunswick, NJ), three stitches of 6-0 polydioxanone at tightness of 0.6N or fibrin glue (Tisseel Duo Quick; Baxter AG, Wien, Austria). Blood sample and pancreas specimens, both at the resection site and far away, were taken 1, 7, and 21 days postoperatively from exposed animals, sham operated animals (n = 18 in each) and from unexposed baseline animals (n = 5). Necrosis, edema, leukocyte infiltration, hemorrhage, vacuolization, and fibrosis were histologically assessed separately., Results: Each resection and sham operation induced similar increase in the amylase activity on day 1 with normalization by day 7. Resection with ultrasonic scissors and electrocautery induced more tissue injury to the pancreas than resection with surgical scalpel independent of the method for hemostasis. The injury, although somewhat milder in intensity, was also observed in parts of the pancreas located far away from the site of resection., Conclusions: Of the compared methods, surgical scalpel resection plus cellulose patch or fibrin glue hemostasis induced the least histological changes in the pancreatic parenchyma. This injury response spread over the pancreas.

Objective: Experimental studies have shown that pancreatic activation of polyamine catabolism occurs during the early stage of acute pancreatitis. Changes in pancreatic polyamines are reflected in the red blood cell (RBC) polyamine contents, correlating with the extent of pancreatic necrosis. The aim of this human study was to examine the changes in polyamine levels in the RBCs of patients with acute pancreatitis., Material and Methods: Twenty-four patients with acute pancreatitis (7 alcoholic, 10 gallstone and 7 of unknown etiology) were recruited in the study. Eighteen patients with non-pancreatic acute abdominal diseases were included as controls, and 6 volunteers were studied as references. Blood samples were collected on admission and during hospitalization to assess polyamine levels. After clinical recovery, the patients revisited the clinic, and RBC polyamine levels were measured again. For comparison, plasma interleukin-6 (IL-6), IL-10 and C-reactive protein (CRP) were measured., Results: In acute pancreatitis patients, there was no difference in RBC polyamine levels on admission compared with those in controls or in volunteers. Putrescine levels on admission were higher in patients with pancreatic necrosis than in patients without necrosis, but there was no difference in spermidine and spermine levels. Patients with pancreatitis of unknown etiology had significantly higher levels of polyamines on admission and throughout hospitalization, but they also had more necrosis, which explained the difference in multivariate analysis. Spermidine and spermine levels increased after clinical recovery. RBC putrescine correlated with IL-6 and IL-10, and spermine correlated with CRP., Conclusions: The results of this study suggest that RBC polyamines change in human acute pancreatitis in several respects, as has been previously observed in experimental pancreatitis.

Aconitum is important in clinical orthopedics. From ancient times to the present day,there were many famous doctors who used this herb to cure many diseases in orthopedics. However, the toxicity always connect with its effect. This will limit its application in clinics. So now the important thing is how to use this herb correctly. This article will give some suggestions about how to use aconitum in orthopedics and to make sure it can be used correctly in future. It has four parts: application in orthopedics, pharmacology, factors related to toxicity and the prospect of the use of aconitum.

Protein transduction domains (PTDs) are small peptides that facilitate the transduction of large molecules such as polyproteins, DNA and viruses into a eukaryotic cell. Here, we demonstrated that a novel PTD (HP4) derived from herring protamine appeared to enter C6Bu1 rat glioma cell lines more rapidly than other known PTDs such as Tat, Antp and Hph-1. Moreover, HP4 significantly enhanced in vitro transduction of recombinant adenoviruses (rAds) into various cancer cell lines, mesenchymal stem cells (MSCs) and dendritic cells, which are relatively resistant to rAd infection. Enhancement of rAd delivery into C6Bu1 and MSCs by HP4 is 20 and 7 times higher than that by Tat, respectively. The increase in the expression of rAd encoding IL-12N220L by HP4 is proportional to its antitumor effect in the ex vivo transduced mouse colon cancer model. Thus, these results suggest that HP4 could be utilized to improve the transduction efficiency of rAd, resulting in enhanced efficacy of rAd-mediated gene therapy, especially for ex vivo-transduced cell therapy.

A novel cytokine interleukin (IL)-23 bears a structural and functional resemblance to IL-12. A recombinant adenovirus expressing IL-23N220L (recombinant replication-defective adenovirus (rAd)/IL-23N220L) that selectively secrets IL-23 was constructed and compared with rAd/IL-12N220L in terms of immunological and antitumor effects. In a prophylactic setting, vaccination with rAd/ovalbumin (OVA) and rAd/IL-23N220L enhanced OVA-specific CD8(+) T-cell responses that were closely associated with complete protection against the subsequent challenge of OVA-expressing E.G7 thymoma. However, in a therapeutic setting, the intratumoral injection of rAd/IL-23N220L showed only marginal antitumor activity against several established tumors such as E.G7, CT26 and B16F10. Interestingly, whereas IL-23 still induced tumor-specific CD8(+) T-cell responses, it could not activate natural killer (NK) cells in vitro and in vivo. In addition, the adoptive transfer of activated NK cells partially restored the therapeutic antitumor effect of IL-23, indicating that NK cells are one of the crucial factors responsible for the regression of established tumors. Taken together, we demonstrated that adenovirus-mediated gene transfer of IL-23 induces a potent prophylactic, but not a therapeutic, antitumor effect.

Osteoarthritis (OA) is a retrogressive arthrosis of which incidence rate increased obviously with the age. Accompany with the aging of the population, the incidence of this disease appeares an upward trend significantly. The etiology and pathogenesis of AO is not very clear at present, so it's important to establish animal models for discussing the pathogenesis of AO and searching reasonable ways to diagnose and treat. The induced animal model is used more commonly, it includes non-operation ways and operation ways, the former includes drug injection, joint immobilization, transarticular impact, etc.; the latter includes destroy the stability of joint, change the stress of joint, snick on condylofemur etc. This article makes a brief review on those questions, looks forward to providing some reference on choosing OA animal model correctly and reasonably.

Background: Stable polyamine homeostasis is important for cell survival and regeneration. Our experimental studies have shown that catabolism of spermidine and spermine to putrescine is associated with the development of pancreatitis. We investigated the pathogenetic role of polyamine catabolism by studying the effect of a methylated polyamine analog on taurodeoxycholate-induced acute experimental pancreatitis., Methods: Acute pancreatitis was induced by infusion of sodium taurodeoxycholate (2%) into the pancreatic duct. Bismethylspermine (Me(2)Spm) was administered as a pretreatment before the induction of pancreatitis or as a treatment after the induction of pancreatitis. The sham operation included laparotomy only. Pancreas tissue and blood were sampled at 24 h and 72 h after the infusion of taurodeoxycholate and studied for pancreatitis severity (serum amylase activity, pancreatic water content, and histology) and polyamine catabolism, which includes spermidine/spermine N(1)-acetyltransferase (SSAT) activity as well as spermidine, spermine, and putrescine concentrations in the pancreas., Results: Sodium taurodeoxycholate-induced acute pancreatitis manifests as increases in serum amylase and pancreatic water content, leukocytosis, and acinar cell necrosis in the pancreas. The activity of SSAT increased significantly together with an increase in the ratios of pancreatic putrescine/spermidine and putrescine/spermine at 24 h, which indicates SSAT-induced polyamine catabolism. Pancreatic water content and necrosis were reduced significantly by the treatment with Me(2)Spm at 24 h but not at 72 h when the polyamine homeostasis had recovered, and the pancreatitis had progressed., Conclusions: Taurodeoxycholate-induced acute pancreatitis was associated with activation of polyamine catabolism in the pancreas. The polyamine analog Me(2)Spm ameliorated the injury in the early stage, but it did not ameliorate the late progression of the pancreatic necrosis at 72 h. Thus, besides proteolytic enzyme activation and the cascades of inflammation, polyamine catabolism may be an important pathogenetic mediator of the early stages of acute pancreatitis.

IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 x 10(4) PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 x 10(8) PFU) caused significant suppression of CD8(+) T cell responses. In addition, the enhancement and suppression of OVA-specific CD8(+) T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8(+) T cell response by IL-12p40 was still observed in IL-12Rbeta2 knockout (IL-12Rbeta2KO), but not in IL-12Rbeta1 knockout (IL-12Rbeta1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rbeta1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.

Objectives: The aim of this study was to investigate biocompatibility and adhesive properties of 6 tissue adhesives available, when applied between the pancreas and jejunum in an experimental model., Methods: Portion of jejunum was glued on the pancreas in rats with 3 cyanoacrylate derivatives (Histoacryl, Dermabond, and Glubran 2), 2 human fibrin sealants (Tisseel Duo Quick and Quixil), and 1 albuminglutaraldehyde sealant (BioGlue). Pancreatic tissue specimens and blood samples were harvested 1, 3, 7, and 21 days after gluing for histological determination and amylase activity measurement. Pancreaticojejunal attachment created with adhesives underwent tensile strength measurement at each time point. Samples were also taken from unoperated rats and sham-operated rats., Results: Exposure and sham groups both induced a similar increase in amylase activity on day 1 with normalization by day 3. Sham operation induced mild changes in the pancreas. Each tissue adhesive induced changes in pancreatic histology to the entire gland. Injurious effect was more severe with the 3 cyanoacrylates than with the 3 fibrin/semisynthetic glues. Histoacryl and Quixil induced lower tensile strength than the other adhesives., Conclusions: All of the tissue adhesives studied induced histological changes in the pancreas of which at least part might be considered harmful. The potentially harmful tissue effects of the preparations tested might compromise the use of these substances in pancreatic surgery.

Background: We have experimental data indicating that the pancreas is easily damaged by any intervention. The present study compared the effects of suture diameter, number of needle passes and suture tightness on rat pancreas., Methods: Under anesthesia, rat pancreas was sutured either with one loose stitch of 6-0 polydioxanone (PDS II) or 3-0 PDS II, with 5 passes of loose running 6-0 PDS II, or with 6-0 PDS II loop tightened to 0.6 or 1.2 N. Amylase activity and pancreatic tissue histology at the suturing site and farther away, were evaluated 1, 3, 7 and 21 days postoperatively., Results: Each suturing exposure and the sham-operation induced temporary amylase activity elevation on day 1 when compared with the baseline. In histology, 3-0 suture, 5 needle passes and 1.2-newton loop induced more damage than 6-0 suture, single needle pass and 0.6-newton loop, respectively. Similar but milder changes were observed in samples from the remote site., Conclusions: The pancreas reacts to suturing with widespread injury response resembling that of acute pancreatitis. In attempting to reduce suturing-induced widespread injury, as few and thin sutures and as loose suture tightness as possible should be used. Although these findings may seem obvious, they have not previously been proven in terms of histology., (Copyright 2008 S. Karger AG, Basel.)

Background/aims: Polyamines are essential to survival, growth, and proliferation of mammalian cells. Previous studies have suggested that the pancreatic polyamine levels may change in acute pancreatitis. In this study, the changes of polyamine levels in the pancreas have been studied with respect to the severity of pancreatitis. We investigated whether there is a relationship in polyamine levels between pancreas and blood, and whether pancreatic and blood polyamine levels change according to the severity of pancreatitis., Methods: In rats, sublethal pancreatitis was induced by intraductal infusion of 2% taurodeoxycholate, while lethal pancreatitis was induced with 6% taurodeoxycholate., Results: Infusion of 6% taurodeoxycholate as compared with 2% resulted in more severe pancreatitis, as revealed by mortality, histology, and serum amylase activity. Pancreatic spermidine/spermine N(1)-acetyltransferase was induced early after pancreatitis and was associated with increased putrescine and decreased spermidine levels. The extent of pancreatic necrosis significantly correlated with the polyamine catabolism indicators pancreatic putrescine/spermidine ratio (r = 0.29, p < 0.01) and pancreatic putrescine/spermine ratio (r = 0.32, p < 0.01). The two pancreatic polyamine ratios correlated well also with the red blood cell polyamine ratios (r = 0.75 and r = 0.72, respectively, both p < 0.01). Furthermore, the extent of pancreatic necrosis correlated with red blood cell putrescine/spermidine (r = 0.32, p < 0.01) and putrescine/spermine (r = 0.37, p < 0.01) ratios., Conclusions: Acute experimental pancreatitis is associated with an early pancreatic spermidine/spermine N(1)-acetyltransferase induction and consequent changes in polyamine levels in pancreas and red blood cells, depending on the severity of pancreatitis. Because changes in red blood cell spermidine, spermine, and putrescine levels evolve already early during the time course of pancreatitis, and correlate with the extent of pancreatic necrosis, their clinical value as early markers of the severity of acute pancreatitis needs to be further evaluated. and IAP., (Copyright 2008 S. Karger AG, Basel.)

Objective: To assess the clinical application of spiral CT 3D reconstruction in forensic diagnosis of rib fracture., Methods: Thirty-five cases of rib fractures were collectively studied by X-ray, regular CT scan, and adoptive 3D reconstruction with spiral CT., Results: Clear and stereoscopic images of rib fractures without interference of the surrounding soft tissue were obtained in all of 35 cases examined., Conclusion: Spiral CT 3D reconstruction could be a valuable means in forensic diagnosis of rib fracture.

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4(+) T cells and stimulating the proliferation of memory CD4(+) T cells. We investigated the pathogenic role of IL-23 in CD4(+) T cells in mice lacking the IL-1R antagonist (IL-1Ra(-/-)), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra(-/-) mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1beta further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4(+) T cells of IL-1Ra(-/-) mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4(+) T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-kappaB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra(-/-) model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.

Immune correlates of protection against hepatitis C virus (HCV) infection are not well understood. Here we investigated 2 naive and 6 immunized chimpanzees before and after intravenous challenge, 12 weeks after the last immunization, with 100 50% chimpanzee infectious doses (CID(50)) of heterologous genotype 1b HCV. Vaccination with recombinant DNA and adenovirus vaccines expressing HCV core, E1E2, and NS3-5 genes induced long-term HCV-specific antibody and T-cell responses and reduced peak viral load about 100 times compared with controls (5.91 +/- 0.38 vs. 3.81 +/- 0.71 logs, respectively). There was a statistically significant inverse correlation between peak viral loads and envelope glycoprotein 2 (E2)-specific antibody responses at the time of challenge. Interestingly, one vaccinee that had sterilizing immunity against slightly heterologous virus generated the highest level of E2-specific total and neutralizing antibody responses as well as strong NS3/NS5-specific T-cell proliferative responses. The other four vaccinees with low levels of E2-specific antibody had about 44-fold reduced peak viral loads but eventually developed persistent infections. In conclusion, vaccine-induced E2-specific antibody plays an important role in prevention from nonhomologous virus infection and may provide new insight into the development of an effective HCV vaccine.

The HIV-1 envelope glycoprotein surface subunit gp120 is an attractive target for molecular intervention. This is because anti-HIV-1 gp120 neutralizing antibodies display the potential ability to inhibit HIV-1 infection. The present investigation describes the construction of a genetically engineered single chain antibody (scFv102) against HIV-1 gp120, its expression and functional evaluation. The parental hybridoma cell line (102) produces an immunoglobulin directed against the conserved CD4-binding region of gp120. cDNAs encoding the variable regions of the heavy (V(H)) and light (V(L)) chains were prepared by reverse transcription PCR and linked together with an oligonucleotide encoding a linker peptide (Gly(4)Ser)(3) to produce a single chain antibody gene. The resulting DNA construct was cloned into a prokaryotic expression vector (pET28) and recombinant scFv102 was expressed in Eserichia coli as an insoluble protein. The denatured scFv102 was refolded and purified by immobilized metal ion affinity chromatography. Purified scFv102 had the same specificity as the intact IgG in immuno-blotting assays and immuno-fluorescence (IF) detection, but ELISA analyses demonstrated the affinity of scFv102 to be 5-fold lower than that of the parental monoclonal antibody. In neutralization assays, scFv102 at concentrations lower than 40 microg/ml exhibited efficient interference with viral replication and inhibition of viral infection (90%) across a range of primary isolates of subtype B HIV-1. These results suggest that the constructed anti-HIV-1 gp120 scFv102 has good biological activity and can potentially be used for in vitro diagnostic and in vivo therapeutic applications.

Anti-HIV-1 gp120 single chain antibody(scFv) gene and staphylococcus extoxin A(SEA) gene were inserted into vector pPIC9K. The recombinant plasmid was integrated into Pichia pastoris by electroporation. High level expression was performed by determining the Muts phenotype and screening muti-copy integrants. The recombinant protein was about 57kD and the production was 50.1 mg/L. It was shown that the two kinds of protein affected the conformation of each other by antibody affinity assay, but the recombinant targeting toxins could highly mediate CTLs to kill HIV-1 target cells.

Interleukin-12 (IL-12) gene was shown to produce both IL-12 and p40 subunit. The excess production of the p40 subunit as a natural antagonist of IL-12 is a major obstacle of IL-12 gene-based cancer therapy. We previously reported that IL-12N220L gene, which selectively reduces the secretion of the p40 subunit, induces long-lasting stronger type 1 helper T cells (T(H)1) and cytotoxic T lymphocyte (CTL) immunity in hepatitis C virus (HCV) E2 DNA vaccination model and higher protection from challenge with tumor cells expressing E2 than IL-12 in a prophylactic setting. Here, we demonstrated that intratumoral injection of IL-12N220L-expressing adenovirus showed better tumor growth inhibition and higher survival rate than that of IL-12 or granulocyte macrophage-colony stimulating factor (GM-CSF)-expressing adenovirus in a therapeutic setting. In particular, the mice cured by IL-12N220L treatment were protected against intravenous rechallenge of the same tumor cells better than those by IL-12 treatment. In addition, the enhanced antitumor activity of IL-12N220L was confirmed in B16F10 lung metastasis model, which correlated with the frequency of tumor-specific interferon (IFN)-gamma-secreting cells. When tested in CT26/NP tumor that expresses influenza nucleoprotein (NP) as a tumor antigen, IL-12N220L induced stronger NP-specific T(H)1 and CTL responses than IL-12, particularly at a later time point, indicating the generating long-term tumor-specific memory T-cell responses. Moreover, the potent antitumor effects of IL-12N220L were further augmented by combination with chemotherapy using farnesyltransferase inhibitor (FTI), LB42908. Taken together, our results suggest that IL-12N220L is superior to IL-12 in cancer immunotherapy, which can be further enhanced by combination with chemotherapy.

Aim: To explore the immune response in mice inoculated with HIV-1 chimeric gene gag-gp120 DNA vaccine and IL-18 plasmid., Methods: BALB/c mice were injected i.m with eukaryotic expression plasmid pVAXIL18 containing IL-18 gene and DNA vaccine plasmid pVAXGE containing HIV-1 gag-gp120 chimeric gene. The specific killing activities of spleen CTL and the titers of serum antibodies of the immunized mice were detected., Results: The specific killing activities of spleen CTL and the titers of serum antibodies in the co-inoculation group were significantly higher than those in the single inoculation group(P<0.05), the vector control group (P<0.01) and PBS control group (P<0.01)., Conclusion: The specific cellular and humoral immunity in mice can be induced by co-inoculating HIV-1 gag-gp120 chimeric gene DNA vaccine and IL-18 plasmid indicating that IL-18 augments immune response as immunoadjuvant.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory infection in young children and the elderly. Studies of mice suggest that RSV suppresses the effector activity of CD8 T cells and the development of pulmonary CD8 T cell memory, in which the impaired effector activity could be recovered by in vitro IL-2 treatment. To investigate the effect of in vivo IL-2 expression on RSV immunity, mice were infected with RSV followed by administration of replication-defective adenovirus expressing IL-2. The effector activity of RSV M2-specific CD8 T cells and the development of CD8 T cell memory in the lung was significantly increased by IL-2 expression. Furthermore, the Ab responses against RSV were augmented by IL-2. Interestingly, weight loss and illness caused by RSV challenge were substantially reduced by IL-2 priming, suggesting that the pathogenesis of RSV-related disease could be prevented by IL-2-mediated enhancement of beneficial immune responses. Thus, our results show that IL-2 has potential to be used as a vaccine adjuvant against RSV infection.

The purification of human immunodeficiency virus type 2 total external glycoprotein gp105 expressed in Pichia pastoris was investigated. Expression conditions were optimized by an orthogonal test. The results from tests of variance analyses showed that the most important parameter for efficient expression of total gp105 in P. pastoris is adequate aeration during methanol induction. The optimum induction conditions for gp105 expression were: more than 85% aeration, induction for 3 days, the initial pH 6.0-7.0 and a final methanol concentration of 1.0%. Under these conditions, the expressed total gp105 was secreted into fermentation broth and reached a yield of 23%, approximately 141 mg/l. Expressed gp105 was isolated and purified by salting out and Sephadex G-100 chromatography and the yield of gp105 was 40%. gp105 was purified to electrophoretic purity and its isoelectric point (pI) was about 5.2 by SDS-PAGE and isoelectrofocusing. The purified gp105 contained approximately 35% carbohydrate, which proved that the expressed gp105 was a glycoprotein. Its N-terminal amino acid was arginine by Dansyl-Cl and the result indicated that expressed gp105 was secreted and cleaved correctly. The results from gp105 ELISA demonstrated that the purified total gp105 showed good reactiongenicity and antigenic specificity.

Interleukin-12 (IL-12), consisting of p40 and p35 subunits, produces both p70 heterodimer and free p40. p70 is essential for the induction of T-helper 1 (Th1) and cytotoxic T-cell (CTL) immunity, whereas p40 inhibits p70-mediated function. Here, we found that mutations introduced into N-glycosylation sites (N220 of murine p40 and N222 of human p40) reduced secretion of p40 but not p70. Co-immunization of N220 mutant mIL-12 gene with hepatitis C virus (HCV) E2 DNA significantly enhanced long-term E2-specific CD8+ T-cell response and protection against tumor challenge compared with that of wild type. Our results indicate that the ratio of p70 to p40 is important for generating sustained long-term cell-mediated immunity. Thus, the mutant IL-12 could be utilized for the development of DNA vaccines as an adjuvant for the generation of long-term memory T-cell responses.

Aim: To study the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) against arteriovenous shunt and middle cerebral artery thrombosis (MCAT) in rats., Methods: Arteriovenous shunt model was adopted to measure thrombus weight; The neurologic deficit (ND) and the infarct size (IS) of the middle cerebral thrombosis (MCAT) model induced by FeCl3 were observed; The effect of ISA on platelet aggregation rate of rat and rabbit by giving ISA in vivo and in vitro was studied., Results: ISA 25, 50, 100 and 200 mg.kg-1 ig was shown to reduced the weight of thrombus in arteriovenous shunt model; ISA 50, 100 and 200 mg.kg-1 ig for 2 times in 24 hours, attenuated the ND of rats subjected to MCAT; ISA 100 and 200 mg.kg-1 reduced IS of rats after MCAT by 27.8% and 31.6%, respectively; ISA 50, 100 and 200 mg.kg-1 ig inhibited platelet aggregation of normal rats; ISA 10(-3)-10(-5) mol.L-1, inhibited rabbit platelet aggregation in vitro., Conclusion: ISA inhibited thrombosis by anti-platelet-aggregation.

To explore a new approach to express HIV-2 external glycoprotein gp105 in methyltrophic yeast, Pichia pastoris, the gp 105 gene was obtained by PCR amplification and was cloned into the secreted expression vector pHILS1. The positive recombinant Pichia pastoris strains were screened and identified by PCR after electroporation and induction by methanol of the gp105 expression. SDS-PAGE and Western blot analyses showed that gp105, unlike gp120 from HIV-1, could be secreted into medium by recombinant Pichia pastoris strain, and the expressed product of a 90 kD glycoprotein showed satisfactory antigenicity with specific antibody.