Chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor for neurotrophic keratopathy

Neurotrophic keratopathy is a persistent defect of the corneal epithelium,with or without stromal ulceration,due to corneal nerve deficiency caused by a variety of etiologies.The treatment options for neurotrophic keratopathy are limited.In this study,an ophthalmic solution was constructed from a chitosan-based thermosensitive hydrogel with long-term release of murine nerve growth factor(CTH-mNGF).Its effectiveness was evaluated in corneal denervation(CD)mice and patients with neurotrophic keratopathy.In the preclinical setting,CTH-mNGF was assessed in a murine corneal denervation model.CTH-mNGF was transparent,thermosensitive,and ensured sustained release of mNGF for over 20 hours on the ocular surface,maintaining the local mNGF concentration around 1300 pg/mL in vivo.Corneal denervation mice treated with CTH-mNGF for 10 days showed a significant increase in corneal nerve area and total corneal nerve length compared with non-treated and CTH treated mice.A subsequent clinical trial of CTH-mNGF was conducted in patients with stage 2 or 3 neurotrophic keratopathy.Patients received topical CTH-mNGF twice daily for 8 weeks.Fluorescein sodium images,Schirmer’s test,intraocular pressure,Cochet-Bonnet corneal perception test,and best corrected visual acuity were evaluated.In total,six patients(total of seven eyes)diagnosed with neurotrophic keratopathy were enrolled.After 8 weeks of CTH-mNGF treatment,all participants showed a decreased area of corneal epithelial defect,as stained by fluorescence.Overall,six out of seven eyes had fluorescence staining scores<5.Moreover,best corrected visual acuity,intraocular pressure,Schirmer’s test and Cochet-Bonnet corneal perception test results showed no significant improvement.An increase in corneal nerve density was observed by in vivo confocal microscopy after 8 weeks of CTH-mNGF treatment in three out of seven eyes.This study demonstrates that CTH-mNGF is transparent,thermosensitive,and has sustained-release properties.Its effectiveness in healing corneal epithelial defects in all eyes with neurotrophic keratopathy suggests CTH-mNGF has promising application prospects in the treatment of neurotrophic keratopathy,being convenient and cost effective.

A novel pulmonary fibrosis murine model with immune-related liver injury

Idiopathic pulmonary fibrosis(IPF),characterized by aggravated alveolar destruc-tion and fibrotic matrix deposition,tendentiously experiences the stage called acute exacerbation IPF(AE-IPF)and progresses to multiple organ damage,especially liver injury.Recent studies have found a variety of immune microenvironment disorders associated with elevated IPF risk and secondary organ injury,whereas current animal models induced with bleomycin(BLM)could not completely reflect the pathologi-cal manifestations of AE-IPF patients in clinic,and the exact underlying mechanisms are not yet fully explored.In the current study,we established an AE-IPF model by tracheal administration of a single dose of BLM and then repeated administrations of lipopolysaccharide in mice.This mouse model successfully recapitulated the clinical features of AE-IPF,including excessive intrapulmonary inflammation and fibrosis and extrapulmonary manifestations,as indicated by significant upregulation of Il6,Tnfa,Il1b,Tgfb,fibronectin,and Col1a1 in both lungs and liver and elevated serum aspartate transaminase and alanine transaminase levels.These effects might be attributed to the regulation of Th17 cells.By sharing this novel murine model,we expect to pro-vide an appropriate experimental platform to investigate the pathogenesis of AE-IPF coupled with liver injury and contribute to the discovery and development of targeted interventions.

Evaluation of antiviral activities of Houttuynia cordata Thunb.extract,quercetin,quercetrin and cinanserin on murine coronavirus and dengue virus infection

Objective:To evaluate the in vitro activities of the ethyl acetate(EA) fraction of Houttuynia cordata(H.cordata) Thunb.(Saururaceae) and three of its constituent flavonoids(quercetin.quercitrin and rutin) against murine coronavirus and dengue virus(DENV).Methods:The antiviral activities of various concentrations of the EA fraction of H.cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus(MHV) and DENV type 2(DENV-2).Cinanserin hydrochloride was also tested against MHV.The EA fraction of H.cordata was tested for acute oral toxicity in C57BL/6 mice.Results:The EA fraction of H.cordata inhibited viral infectivity up to 6 d.Cinanserin hydrochloride was able to inhibit MHV for only 2 d.The 50%inhibitory concentrations(IC_(50)) of the EA fraction of H.cordata added before the viral adsorption stage were 0.98 μg/mL for MHV and 7.50 μg/mL for DENV-2with absence of cytotoxicity.The mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute toxicity,with normal histological features of major organs.Certain flavonoids exhibited comparatively weaker antiviral activity,notably quercetin which could inhibit both MHV and DENV-2.This was followed by quercitrin which could inhibit DENV-2but not MHV,whereas rutin did not exert any inhibitory effect on either virus.When quercetin was combined with quercitrin,enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed.However,the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction.Conclusions:The compounds in H.cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vim.H.cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.

Two stomach-originated lactobacillus strains improve Helicobacter pylori infected murine gastritis

AIM:To investigate the potential anti-Helicobacter pylori(H.pylori ) and anti-inflammation in vivo effects of two lactobacillus strains from human stomach.METHODS:Forty H.pylori infected Balb/c mice were randomly divided into 4 groups:proton pump inhibitor and antibiotics triple treated group,Lactobacillus fermenti(L.fermenti ) treated group,Lactobacillus acidophilus treated group and normal saline control group.Ten uninfected mice were also included as blank control group.The infection of H.pylori was detected by rapid urease tests,Giemsa staining and bacterial culture.The colonization of H.pylori was assessed in bacterial density score and gastric inflammation was assessed in histological score.The colonization of L.fermenti was performed by fluorescent probe.RESULTS:Histopathologic evaluation showed significant release of mucosal inflammation in gastric antrum and gastric body in lactobacillus treated groups and triple treated group.H.pylori eradication rate in both lactobacillus treated groups and triple treated group were higher than normal saline control group.Lactobacillus treated groups and triple treated group showed significant decrease of H.pylori bacterial density.CONCLUSION:Both lactobacillus strains have a significant anti-H.pylori activity;L.fermenti displays more efficient antagonistic activity in vivo against H.pylori infection.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.

N-acetylcysteine and glycyrrhizin combination:Benefit outcome in a murine model of acetaminophen-induced liver failure

BACKGROUND Acetaminophen overdose is the most frequent cause of drug-induced liver failure in developed countries.Substantial progress has been made in understanding the mechanism of hepatocellular injury,but N-acetylcysteine remains the only effective treatment despite its short therapeutic window.Thus,other hepatoprotective drugs are needed for the delayed treatment of acetaminopheninduced hepatotoxicity.Our interest focused on glycyrrhizin for its role as an inhibitor of high mobility group box 1(HMGB1)protein,a member of the family of damage-associated molecular pattern,known to play an important pathological role in various diseases.AIM To investigate the efficacy of the N-acetylcysteine/glycyrrhizin combination compared to N-acetylcysteine alone in the prevention of liver toxicity.METHODS Eight-week-old C57BL/6J wild-type female mice were used for all our experiments.Mice fasted for 15 h were treated with acetaminophen(500 mg/kg)or vehicle(phosphate-buffered saline)by intraperitoneal injection and separated into the following groups:Glycyrrhizin(200 mg/kg);N-acetylcysteine(150 mg/kg);and N-acetylcysteine/glycyrrhizin.In all groups,mice were sacrificed 12 h following acetaminophen administration.The assessment of hepatotoxicity was performed by measuring plasma levels of alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase.Hepatotoxicity was also evaluated by histological examination of hematoxylin and eosin-stained tissues sections.Survival rates were compared between various groups using Kaplan-Meier curves.RESULTS Consistent with data published in the literature,we confirmed that intraperitoneal administration of acetaminophen(500 mg/kg)in mice induced severe liver injury as evidenced by increases in alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase but also by liver necrosis score.Glycyrrhizin administration was shown to reduce the release of HMGB1 and significantly decreased the severity of liver injury.Thus,the co-administration of glycyrrhizin and N-acetylcysteine was investigated.Administered concomitantly with acetaminophen,the combination significantly reduced the severity of liver injury.Delayed administration of the combination of drugs,2 h or 6 h after acetaminophen,also induced a significant decrease in hepatocyte necrosis compared to mice treated with N-acetylcysteine alone.In addition,administration of N-acetylcysteine/glycyrrhizin combination was associated with an improved survival rate compared to mice treated with only N-acetylcysteine.CONCLUSION We demonstrate that,compared to N-acetylcysteine alone,co-administration of glycyrrhizin decreases the liver necrosis score and improves survival in a murine model of acetaminophen-induced liver injury.Our study opens a potential new therapeutic pathway in the prevention of acetaminophen hepatotoxicity.

Murine models based on acute myeloid leukemia-initiating stem cells xenografting

Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.

Great efficacy of sulfachloropyrazine-sodium against acute murine toxoplasmosis

Objective:To identify more effective and less toxic drugs to treat animal toxoplasmosis.Methods:Efficucy of seven kinds of sulfonamides against Toxoplasma gondii(T.gondii)in an acute murine model was evaluated.The mice used throughout the study were randomly assigned to many groups(10 mice each),which either remained uninfected or were infected intraperitoneally with lachyzoites of T.gondii(strains RH and CN).All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates.Sulfadiazine-sodium(SD)was used for comparison.Results:The oplimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium(SPZ)for five days.Using this protocol,the average survival time and the time-point of 50%fatalities were prolonged significantly compared with SD treatment.Treatment with SFZ protected 40%of mice from death,and the heart and kidney tissue of these animals was parasite-free,as determined by nestedPCK.SPZ showed excellent therapeutic effects in the treatment of T.gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T.gondii in animals.Conclusions:It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis.

Therapeutic effect of intravesical or intratumoral injection of oncolysate transfected with recombinant vaccinia virus encoding human IL-2 gene on murine melanoma model

Oncolysate, a debris of tumor cells, has been provento be effective in tumor active immunotherapy, it wasreported that the vaccinia virus, especially recombinantvaccinia viruses encoding human IL-2 (rVV-IL-2 ),enhanced the immunogenicity of transfected tumor cells.In this experiment, the murine melanoma cell B16-F10oncolysates trans fected by rVV-IL-2 (IL-2VBO) wereused as vaccine. The IL-2VBO or TK-VBO was preparedby incubating B16-F10 cells with rVV-IL-2 or rVV-TK

A ModelSystem of Prim ary Murine Hepatocytes Infected byMurine Cytom egalovirus

In order to establish a model system of the murine hepatocyte infection by murine cytomegalovirus (MCMV), the primary cultured murine hepatocytes were obtained in a modified low serum medium system by a non perfusion method, and then infected by Smith strain MCMV. Infected hepatocytes showed characteristic cytopathic effect (CPE) at 30 h after infection, in which a large number of viral particles was found and ultrastructures were destroyed (as revealed by disappearance of bile canalicula and organelles) under the electron microscope and MCMV immediate early genes were detected by in situ hybridization. Meanwhile, infected cells produced albumin significantly less than corresponding uninfected controls. On the contrary, uninfected controls simultaneously cultured under the same conditions showed normal function and ultratructure (glycogen rosettes, bile canalicula, wheel like mitochondria and well developed rough and smooth endoplasmic reticula). These results demonstrated that a model system of primary cultured murine hepatocytes infected by MCMV was successfully set up.

Cytokine profile in murine toxoplasmosis

Objective:To investigate which cytokines are produced after acute infection of mice with Toxoplasma gondii(T.Gondii) RH strain.Methods:Mus domesticus domesticus mice in infected group were inoculated with with highly virulent T.Gondii RH strain by intraperitoneally.Serum samples were obtained from infected and non-infected mice for cytokine levels for ELISA assay.Results:The concentrations of tumor necrosis factor-α,interferon-γ,interleukin(IL)- 10 and IL-12 in the cardiac blood sample of the infected mice were significantly higher than those in uninfected controls(P【0.05).The levels of transforming growth factor-1βdecreased in mice infected with T.gondii compared to those of the controls,the decrease was statistically significant(P【0.05).No significant difference was observed in levels of IL-4 between infected and healty control groups(P】0.05).Conclusions:According to our findings,immune response into T helper type 1 was predominant during acute T.gondii infection.Further characterization and purification of Toxoplasma molecule(s) implicated in the regulation of cytokines could lead to the development of new drug prospects to control Toxoplasma infection.

The effect of inhibitor peptide for ADAM8 in attenuating ovalbumin-induced murine asthma

Targeted peptides have been identified as showing great promise for treatment of various diseases including asthma.Asthma is considered of difficuIt-to-treat due to its unclear etiology,thus usually requiring life-long treatment.Current strategies for asthma therapy are hampered with undesirable side effects,poor targeting and failure in modulating airway hyperresponsiveness,leading to pressing need of developing more targeted and effective therapeutic sites for asthma.Recently,a disintegrin and metalloproteinase 8(ADAM8)have been shown to over-

ESTABLISHMENT OF A MURINE ASCITES HEPATOMA CELL LINE H_(22)-F_(25)/L AND ITS BIOLOGICAL CHARACTERISTICS

Having been passed for 160 generations, a cell linedesignated as H22-F25/L was established from a murine tumorlymphatic metastatlc model H22-F25 which had been set up in our college. The cell line was in suspension culture with a rapid proliferation and stable growth. The peak tune of cell division and proliferation was 48 and 96 hours after culture. In a week, the cell number was Increased by 25 tunes. H22-F25/L still keeps the features of a poorly differentiated cancer. Its tumor inducing rate (in vivo)was 100% in 615 mice. Lymph node metastasis rate was 50% and pulmonary metastasis rate 10%. H22- F25/ L Is a population of heterogenetlc tumor cells Including 2 stem cell lines (the model number of chromosomes being 43 in 40% tumor cells and 86 in 32%) and some side lines. The common marker chromosomes M1, M2, M3 and M4 were present in all stem and side lines.

In vivo gene therapy of murine melanoma mediated by recombinant vaccinia virus encoding human IL-2 gene

Direct gene transfer into somatic tissue in vivo is adeveloping technology with potential application forcancer gene therapy. Retrovirus vector, which was aneffective vehicle, still has some disadvantages ingenerating high titer recombinant vectors andmanipulating to mediate in viro gene transfer. In thispaper, recombinant vaccinia virus vector encoding

Antitumor effects of cytokine gene-encoded vaccinia virus on murine melanoma

Recombinant vaccinia virus has many advantagesover more restricted vectors like retrovirus andadenovirus. The proven safety of vaccinia virus, which isrestricted to local and transitory infection, favors clinicalapplication of vaccinia virus to deliver cytokines locally.

Combination of suicide gene and interleukin 2 gene transfer elicited potent antitumor effects on murine melanoma

Antitumor effects of combined transfer of suicidegene and cytokine gene were investigated in this report.Adenovirus harboring E.coli. cytosine deaminase (CD)gene (Ad-CD) and murine interleukin 2 (IL-2) gene(Ad-IL-2) were used for gene transfer in vitro and invivo. C57BL/6 mice were inoculated subcutaneously withB16F10 melanoma cells and three days later treated withadenovirus injection at the site of tumor inoculation.Significant inhibition of tumor growth was achieved

LPS-induced activation of phospholipase A_2 phospholipase Cand protein kinase C of murine macrophage-like cell lines （J774 and P388D1）

A murine macrophage-like cell line,J774,acquried,in response to LPS,an ability to kill tumor necrosisfactor（TNF）-insensitive target P815 mastocytoma cells,whereas another cell line,P388D1,did not.LPS-triggered signaling mechanisms between the two celllines were compared with an aim to inquire about thepossible nature of the above-mentioned difference.Theresults showed that two cell lines respond to LPS-treatment by parallel activation of both phospholipasesC and A2（PLC and PLA2）to approximately the sameextent.The maximum response of both enzymes of J774cells was noted within 10 min of the treatment,whereas that of P388D1 cells required more than 20min.The other properties of LPS-responsive enzymesstudied were similar between two cell lines,ineludingActivation of PLC and PLA2 and PKC in macrophages by LPSCa2+augmentation of enzyme activation,participationof guanine nucleotide binding （G） proteins in theinitial activation processes,and inhibition of enzymeactivation by the prior treatment of cells with choleraorpartussis toxins etc.Moreover,LPS-triggered activationof PLC and PLA2 was found to be followed by theincrease of PKC activities in both cell lines.In spite ofthese similarities,J774 cells possessed both basic andacidic forms of PKC activities,while P 388D1 cells ownedonly PKC of basic form.Nevertheless,the question whyJ774 cells,but not P388D1 cells,can acquire thetumoricidal actiyity,aganist P815 cells following LPS-treatment remains to be answered.

The effects on biological behaviors of P815 murine mastocytoma cell line induced by the transduction of mouse interleukin-2 gene

The whole coding region including signal peptidesequence of mouse interleukin-2 (mIL-2) cDNA wasobtained by PCR method and then cloned into the propersites of pUC18 vector. Sequencing analysis throughSanger method (dideoxy-mediated chain-terminationmethod) proved the consistency of mIL-2 cDNAsequence with that reported before. The fragment

Inoculation with IL-2 and B7-1 co-transfected G422 murine glioblastoma cells resulting in enhanced antitumor immunity in vivo

It’s well known that interleukin-2 (IL-2) plays animportant role in eliciting antitumor immunityparticularly mediated by T cells. In addition, theexpression of MHC can be enhanced by IL-2 genetransfection in tumor cells. Recent studies have indicatedthat at least two signals are required for the activation ofnaive T cells by antigen-bearing target cells: an antigen-sopific signal and a crystimulatory signal. B7-l is