Ml Of PBS Research Articles

Lactobacillus plantarum BSGP201683 Isolated from Giant Panda Feces Attenuated Inflammation and Improved Gut Microflora in Mice Challenged with Enterotoxigenic Escherichia coli.

In this work, we searched for an effective probiotic that can help control intestinal infection, particularly enterotoxigenic Escherichia coli K88 (ETEC) invasion, in giant panda (Ailuropoda melanoleuca). As a potential probiotic strain, Lactobacillus plantarum BSGP201683 (L. plantarum G83) was isolated from the feces of giant panda and proven beneficial in vitro. This study was aimed to evaluate the protective effect of L. plantarum G83 in mice challenged with ETEC. The mice were orally administered with 0.2 mL of PBS containing L. plantarum G83 at 0 colony-forming units (cfu) mL−1 (control; negative control, ETEC group), 5.0 × 108 cfu mL−1 (LDLP), 5.0 × 109 cfu mL−1 (MDLP), and 5.0 × 1010 cfu mL−1 (HDLP) for 14 consecutive days. At day 15, the mice (LDLP, MDLP, HDLP, and ETEC groups) were challenged with ETEC and assessed at 0, 24, and 144 h. Animal health status; chemical and biological intestinal barriers; and body weight were measured. Results showed that L. plantarum G83 supplementation protected the mouse gut mainly by attenuating inflammation and improving the gut microflora. Most indices significantly changed at 24 h after challenge compared to those at 0 and 144 h. All treatment groups showed inhibited plasma diamine oxidase activity and D-lactate concentration. Tight-junction protein expression was down-regulated, and interleukin (IL)-1β, IL-6, IL-8, TLR4, and MyD88 levels were up-regulated in the jejunum in the LDLP and MDLP groups. The number of the Enterobacteriaceae family and the heat-labile enterotoxin (LT) gene decreased (P < 0.05) in the colons in the LDLP and MDLP groups. All data indicated that L. plantarum G83 could attenuate acute intestinal inflammation caused by ETEC infection, and the low and intermediate doses were superior to the high dose. These findings suggested that L. plantarum G83 may serve as a protective probiotic for intestinal disease and merits further investigation.

ID: 1006 Allogeneic adipose -derived mesenchymal stem cell transplantion enhanced the expression of angiogenic factors on mouse acute hindlimb ischemia model

The cell migration and/or molecular mechanism in healing of damaged vascular or muscle tissue are emerging field with strong research interest worldwide. This study focuses on evaluating the role of allogenic adipose-derived mesenchymal stem cells (ADMSCs) in restoring damaged tissue through the migration of cells and the expression of genes related to myocyte regeneration and angiogenesis on hindlimb ischemic mouse model. ADMSCs were labeled with GFP (ADMSC-GFP). The proximal end of the femoral blood vessel of mice (over 6 months old) was ligated at 2 positions then cut between the two ties. Hindlimb ischemic mice were randomly divided to 2 groups. Group 1 (n=36) was injected PBS (100 μl), group 2 (n=36) was transplanted ADMSC-GFP (106 cell/100 μl PBS) near the cutting position. The migration of ADMSC-GFP in hindlimb was analyzed by UVVis system. The expression of genes related to angiogenesis and muscle tissue repairing was quantified by real time RT-PCR. Muscle tissue structure was histological analyzed by H&amp;E staining. The result showed that ADMSC existed in grafted hindlimb during 7 days. Grafted cells migrated to other damaged areas such as thighs and heels. Ischemic hindlimb were stimulated to increasing expression of several angiogeneic genes including Flt-1, Flk-1 Ang-2 in both group. Especially, the expression of Ang-2 and myogenic-related gene MyoD were significantly increased in ADMSC-treated group compared to non-treated group; and increased at 28th day compared to 3rd day. The other factors such as HGF, CD31, Myf5, TGF-β also expressed much more in ADMSC treated group than the another one. Muscle histological structure in ADMSC group were siminar the normal mouse after 28 day. Thus, grafted-ADMSC was able to migrate to other area in injured hindlimb, existed for about 7 days and have significant positive effects on stimulating the expression of myogenic and angiogenesis- related genes.

MRI evaluation of frequent complications after intra-arterial transplantation of mesenchymal stem cells in rats

Intra-arterial transplantation of mesenchymal stem cells (MSCs) is an effective delivery route for treatment of ischemic brain injury. Despite significant therapeutic effects and targeted cells delivery to the brain infraction, serious adverse events such as cerebral embolism have been reported and may restrict potential clinical applications of this method. In current study, we evaluate potential complications of intra-arterial MSCs administration and determine the optimum parameters for cell transplantation. We injected SPIO-labeled human MSCs via internal carotid artery with different infusion parameters and cell dose in intact rats and in rats with the middle cerebral occlusion stroke model. Cerebrovascular complications and labeled cells were visualized in vivo using MRI. We have shown that the incidence of cerebral embolic events depends on such parameters as cell dose, infusion rate and maintenance of blood flow in the internal carotid artery (ICA). Optimal parameters were considered to be 5×105 hMSC in 1 ml of PBS by syringe pump with velocity 100 μ/min and maintenance of blood flow in the ICA. Obtained data should be considered before planning experiments in rats and, potentially, can help in planning clinical trials in stroke patients.

AB053. Labeling of prostate tumor-specific replication-selective oncolytic adenoviruses with radioactive 125I: inhibitory effects on prostate cancer cell

BackgroundThe authors established a 125I-labeled replication-selective oncolytic adenovirus and human telomerase reverse transcriptase/prostate-specific antigen (125I-RSOAds-hTERT/PSA) oncolytic adenovirus marker and investigated the effects of different labeling conditions. This study also explored the possible mechanism whereby 125I-RSOAds-hTERT/PSA inhibited the proliferation of prostate cancer cells.MethodsN-bromosuccinimide (NBS) was used as an oxidant for 125I labeling, and various concentrations of oncolytic viruses and NBS were prepared to determine the optimal conditions for labeling. The effects of the amount of 125I, reaction time, pH, and reaction volume on the labeling rate of the 125I-RSOAds-hTERT/PSA oncolytic adenovirus marker were measured. Radioactive oncolytic adenoviruses were isolated and purified by column chromatography; the radiochemical purities of the 125I-RSOAds-hTERT/PSA marker at different times were detected by paper chromatography. After the addition of radioactive iodine-labeled prostate cancer-specific oncolytic adenoviruses to prostate cancer cells, changes in the inhibitory rate were measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays.ResultsThe radiochemical purity of the 125I-RSOAds-hTERT/PSA marker was >95%, and the marker was stable (93–94%) after storage at 4 °C for 7 days. The optimal conditions were 0.5 µL of 125I (about 0.2 mCi, 7.4 MBq), 25 µg of NBS, 100 µL of 8×109 viral protein (VP)/mL 125I-RSOAds-hTERT/PSA virus solution, 3 min of reaction time, pH 7.5, and 120 µL PBS. Radioactive iodine-labeled prostate cancer-specific oncolytic adenoviruses inhibited the proliferation of prostate cancer cells significantly.ConclusionsRadioactive 125I labeling of the hTERT/PSA dual-regulated prostate cancer-specific oncolytic adenovirus is feasible, and the radiochemical purity of the marker was stable under defined conditions. Radioactive iodine-labeled prostate cancer-specific replication-selective oncolytic adenoviruses significantly inhibited prostate cell growth.

Establishment of an experimental glaucoma animal model: A comparison of microbead injection with or without hydroxypropyl methylcellulose.

The present study aimed to compare microbead injection with and without hydroxypropyl methylcellulose (HPM) in order to establish an experimental animal model of glaucoma. This model was established in C57BL/6 mice and transgenic mice expressing cyan fluorescent protein (CFP) under the control of the Thy1 promoter in retinal ganglion cells (RGCs). C57BL/6 mice aged between 12 and 20 weeks old were randomly separated into three groups, which received different injections into the anterior chamber of the eye. Group A (microbead) received 2 µl microbeads (10×106 beads/ml) and 1 µl air. Group B (microbeads + HPM) received 2 µl microbeads and 1 µl HPM. Group C (control group) received 2 µl PBS and 1 µl air. The intraocular pressure (IOP) was measured with a tonometer under topical anesthesia daily for 1 month. A single injection of microbeads, with or without HPM, induced consistent IOP elevation when compared with the control group. Thy1-CFP mice received an injection of 2 µl microbeads and 1 µl HPM into the anterior chamber of the eyes, and the number of CFP+ RGCs was subsequently assessed in vivo by confocal scanning laser microscopy in the same area of the retina weekly for 6 weeks. The results from in vivo imaging of Thy1-CFP mice were comparable with the immunohistochemical staining results from the C57BL/6 mice. The combined injection of microbeads and HPM induced longer and higher peaks of IOP elevation when compared with the microbeads alone. The rate of RGC loss following the administration of microbeads alone was 25.0±1.3% 6 weeks after the initial IOP elevation, while it was 33.2±1.9% following the administration of microbeads + HPM. These results indicate that the injection of microbeads + HPM is a more effective method of establishing a mouse model with chronic elevation of IOP. In addition, the in vivo imaging that can be used with this technique provides an effective and noninvasive approach for monitoring the progress of RGC loss.

Abstract 168: Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling

Abstract The aim of this study is to determine the effect of the tissue inhibitor of metalloproteinase-2 (TIMP-2) on lung tumorigenesis and investigate the underlying molecular mechanisms using an orthotopic mouse model with a loss of function mutation in the Timp2 gene (T2M). T2M and wildtype (WT) control mice were given 1x106 Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) in 50μL PBS via intratracheal installation. IVIS imaging revealed a higher tumor burden in T2M mice compared to WT littermates, suggesting that loss of function of the Timp2 gene enhances tumor growth (p&amp;lt;0.05). We also conducted a Kaplan-Meier analysis to determine the effect of this mutation on mortality following cancer development. We found that LL/2 tumor-induced mortality was significantly higher in T2M mice compared to WT controls (p = 0.01). Histologic analysis and H&amp;E staining of lung tissue sections revealed a significant increase in the number of tumor nodules of T2M mice compared to WT controls (p &amp;lt;0.01). CD31 staining showed a significant increase in microvessel density (MVD) in T2M mice compared to WT controls (p&amp;lt;0.01). Given that VEGF is a primary driver of tumor neovascularization, we determined mRNA levels of VEGF expression in healthy and tumor bearing mice by qRTPCR analysis. Interestingly, basal VEGF expression levels were increased in lungs of both non-tumor bearing and tumor bearing T2M mice compared to WT controls (p&amp;lt;0.01). VEGF is the direct downstream target of HIF-2α, a transcription factor implicated in tumor hypoxia and expression correlates with decreased overall survival in non-small cell lung cancer (NSCLC) patients. Similarly, an examination of TIMP-2 and HIF-2α mRNA expression in a small cohort of patient samples revealed decreased TIMP-2 and increased HIF2α in NSCLC tumors compared to surrounding non-tumor lung tissue. Hypoxia induces EGFR signaling through HIF-2α. Thus, we determined the effect of mutated T2M on the EGFR signaling pathway. We found increased levels of EGFR phosphorylation as well as downstream ERK and Akt activation in tumor bearing and non-tumor bearing T2M lungs compared to WT controls. In conclusion, these findings offer new avenues for TIMP-2 research in regulating hypoxic mediators within the tumor microenvironment through EGFR signaling, suggesting TIMP-2 as a novel bio-therapeutic for lung cancer therapy. * Corresponding author This work is supported by center for cancer research, intramural research program, NCI/NIH research grants ZIA BC011204 &amp; ZIA SC 009179 to WGSS Citation Format: Sarvesh Kumar, Sandra Jensen, David Peeney, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. Loss of function mutation in TIMP2 gene accelerates tumorigenesis and mortality in murine model of lung cancer through EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 168. doi:10.1158/1538-7445.AM2017-168

Abstract 2225: Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors

Abstract Green tea polyphenols (GTP) and its major constituent, epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells that reduces invasiveness and migration capabilities; however, the mechanisms whereby these effects occur are not well understood. RECK, a novel tumor suppressor and the tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) genes negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor invasion, angiogenesis and metastasis. In the present study, we demonstrate that GTP mediate epigenetic induction of RECK and TIMP-3 thereby playing a key role in suppressing invasiveness and gelatinolytic activity of MMP-2 and MMP-9 in vivo in an orthotopic implantation model of human prostate cancer. Athymic nude mice were implanted with human prostate cancer LNCaP tumor in the ventral prostate for 2 weeks and later treated DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (AZA), histone deacetylase inhibitor, trichostatin A (TSA) and histone methyltransferase inhibitor, 3-Deazaneplanocin A (DZNep) individually at 0.1 mg/kg body weight intraperitoneally at alternate days/week; and combination of AZA+TSA and DZNep+TSA at similar doses and times; whereas GTP was provided peroral by gavage at 7.5 and 15.0 mg/kg body weight freshly prepared in 100µl PBS. Treatment of mice with GTP resulted in marked decrease in tumor growth and its local invasion in dose dependent manner, compared to treatment with epigenetic inhibitors and their combination after 8 weeks of intervention. GTP treatment significantly reduced serum levels of MMP-2, MMP-9 and VEGF, compared to treatment with epigenetic inhibitors alone. Combination of AZA+TSA exhibited similar effect which was equivalent to the lower dose of GTP treatment. Furthermore, GTP treatment significantly reduced EZH2 and H3K27me3 and class I HDAC protein levels in tumors. GTP partially reversed the hypermethylation status of RECK and TIMP3 gene and significantly enhanced their protein expression in the tumor tissue. Inhibition of VEGF, MMP-2 and MMP-9 levels were also noted after GTP treatment in tumor tissue in dose-dependent manner. Our findings suggest that induction of RECK and TIMP-3 are key epigenetic events modulated by GTP that results in suppression of matrix degradation and angiogenesis to delay prostate cancer invasion and its subsequent progression. Citation Format: Eswar Shankar, Natarajan Bhaskaran, Rajnee Kanwal, Sanjay Gupta. Green tea polyphenols suppress tumor growth and invasion by targeting matrix metalloproteinases, RECK and TIMP-3, in a mouse model implanted with prostate tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2225. doi:10.1158/1538-7445.AM2017-2225

Abstract 1701: The novel anti-CD205 antibody drug conjugate (ADC) MEN1309 shows strong antitumoral activity in diffuse large B cell lymphoma (DLBCL)

Abstract Background. Up to one third of DLBCL patients still succumb to their disease: novel therapeutic approaches are needed. MEN1309 is a novel ADC consisting of an anti-CD205 monoclonal antibody conjugated to the DM4 maytansine derivative through a cleavable linker. Here, we assessed its anti-DLBCL antitumoral activity. Methods 24 DLBCL cell lines were exposed to MEN1309 and, as control, to IgG-conjugated DM4 for 72h. Cell proliferation was measured with MTT. Apoptosis activation, defined by at least 1.5 fold increase in the caspase 3/7 signal respect to controls, was measured with the Promega ApoTox-Glo Triplex Assay. Xenografts (15 x 106 cells/mouse, 200 μL of PBS) were established s.c. into the left flanks of female NOD-SCID mice; i.v. treatments started with tumors of 250-350 mm3 volume. Results. MEN1309 showed strong cytotoxic activity in DLBCL (median IC50 = 300 pM; 95%CI, 200-771), while the IgG-conjugated DM4 toxin was 100x less active (30 nM; 95%CI, 20-33). MEN1309 induced apoptosis in 17/24 (71%) DLBCL. No difference was seen based upon DLBCL cell of origin, MYC or BCL2 translocations or TP53 status. MEN1309 activity was highly correlated with its target expression with an inverse correlation between IC50 values and CD205 expression at flow cytometry or RT-PCR (R = - 0.79, P &amp;lt; 0.0001). No correlation was seen between CD205 expression and IgG-conjugated DM4 activity. MEN1309 anti-tumor activity was in vivo confirmed in a DLBCL model (OCI-Ly10) characterized by high CD205 expression. MEN1309 was administered at 3 different doses: 1.25, 2.5 and 5 mg/Kg once / 3 weeks. Groups of control treated with IgG-DM4 (5 mg/Kg, every 3 weeks) were also used. MEN1309 5mg/Kg eradicated tumors in all mice with a single dose, as also shown by highly significant differences versus control mice (D7, D21, D28; P &amp;lt; 0.01). While all other groups had to be stopped by D35, the MEN1309 5mg/Kg group skipped the second treatment at D21 because there was no palpable tumor, and mice resulted cured even 2 months later (Kaplan-Meier, survival analysis, P &amp;lt; 0.0001). MEN1309 2.5 mg/Kg delayed tumor growth versus control (D21, P 0.039). Much lower activity was observed with MEN1309 1.25 mg/Kg (D7, P 0.012) and with IgG-DM4 (D21, P 0.049; D28, P 0.046). Conclusions. In DLBCL models, the novel ADC MEN1309 had strong in vitro and in vivo anti-tumor activity, which is highly correlated with the expression of its target. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Francesca Guidetti, Maurilio Ponzoni, Roberta Pittau Bordone, Alessio Fiascarelli, Andrea Rinaldi, Ivo Kwee, Afua Adjeiwaa Mensah, Anastasios Stathis, Davide Rossi, Georg Stussi, Emanuele Zucca, Giuseppe Merlino, Mario Bigioni, Monica Binaschi, Francesco Bertoni. The novel anti-CD205 antibody drug conjugate (ADC) MEN1309 shows strong antitumoral activity in diffuse large B cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1701. doi:10.1158/1538-7445.AM2017-1701

Abstract 4182: The two novel BTK-inhibitors M2951 and M7583 show in vivo anti-tumor activity in pre-clinical models of B cell lymphoma

Abstract Background. Diffuse large B cell lymphoma of the activated B cell like type (ABC DLBCL) is an aggressive lymphoma in which the inhibition of the Bruton’s tyrosine kinase (BTK) has shown biologic and clinical relevance. M7583 and M2951 are two novel highly selective irreversible BTK inhibitors (Fest C et al, 2016). M7583 has shown activity in a mouse model of lupus erythematosus (Bender et al, 2016). Here, we evaluated the anti-tumor activity of M2951 and M7583 in an in vivo ABC DLBCL model. Methods. Xenografts were established by s.c. injecting TMD8 lymphoma cells (100 µL of PBS, 107 cells/mouse) into the left flanks of female NOD-SCID mice (6 weeks of age and around 20g of body weight). Treatments (QD, po) were started with an average tumor volume of 100mm3 on groups of 10 mice each. Results. NOD-SCID mice engrafted with the ABC DLBCL TMD8 were treated with three different concentrations of M7583 (1 mg/Kg; 3 mg/Kg; 10mg/Kg) or M2951 (5 mg/Kg; 15 mg/Kg; 25mg/Kg) or vehicle, as control group. M2951 slowed tumor growth at all doses starting after 10 days of treatment. M7583 at 3 and 10 mg/Kg showed stronger anti-tumor activity than M2951, already evident after 5 days of treatment (P&amp;lt;0.05) and improving with time (after 10 days, P&amp;lt; 0.005). Moreover, while most groups had to be stopped after 2 weeks of treatment (D30 after engraftment) because of the reaching of the maximum tumor volume, allowed by the ethical veterinary committee, mice treated with M7583 at the two highest doses were able to continue treatment for additional six (3 mg/Kg) or ten days (10mg/Kg), when tumors became bigger than 2000 mm3, indicating a high activity of M7583. No body weight losses and sign of toxicity were detected. Conclusion. The two novel BTK inhibitors M2951 and M7583 showed promising in vivo anti-tumor activity in an ABC-DLBCL model. These data provide the rational for further investigating these compounds. Citation Format: Eugenio Gaudio, Chiara Tarantelli, Emanuele Zucca, Davide Rossi, Anastasios Stathis, Francesco Bertoni. The two novel BTK-inhibitors M2951 and M7583 show in vivo anti-tumor activity in pre-clinical models of B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4182. doi:10.1158/1538-7445.AM2017-4182

Effects of intranasal treatment with slow (GYY4137) and rapid (NaHS) donors of hydrogen sulfide in lipopolysaccharide-induced airway inflammation in mice

We have investigated the effects of slow (GYY4137) and rapid (NaHS) hydrogen sulfide (H2S) releasing donors in lipopolysaccharide (LPS)-induced airway inflammation in mice. LPS (0.1 mg/ml) in 60 μl PBS was administered by the intranasal (i.n.) route and control group received vehicle, whereas the subgroups of mice were treated with i.n. GYY4137 or NaHS. The tracheal reactivity, inflammatory cell count in bronchoalveolar lavage (BAL) fluid and lung histopathology were evaluated in all groups 48 h after LPS/PBS applications. 5-Hydroxytryptamine (5-HT)-induced contraction response in isolated tracheas was enhanced after LPS treatment but carbachol response was not altered. Incubation with atropine (10−6 M), 5-HT2A receptor antagonist ketanserin (10−9–10−7 M) and 5-HT3 receptor antagonist alosetron (10−8 and 10−7 M) prevented 5-HT-induced hyperreactivity whereas 5-HT4 receptor antagonist GR113808 (10−7 M, 10−6 M) did not have any effect in LPS-treated group. Electrical field stimulation (EFS) of isolated tracheas elicited frequency-dependent contractile response, which was not altered by LPS treatment alone but was enhanced in the presence of 5-HT (10−9-10−4 M). This data indicated that 5-HT2A and 5-HT3 receptors, and acetylcholine released from cholinergic nerves were contributing to 5-HT-induced hyperreactivity in the present experiments. The increase in neutrophil count along with cytokine (IL-1β, TNF-α) levels in bronchoalveolar lavage (BAL) fluid and histopathological changes like paranchymal inflammation and interalveolar thickening were determined in LPS-treated mice. H2S production in lung homogenates were determined by the methylene blue assay, and found to be similar in both LPS and control groups. The experiments conducted after i.n. treatment with H2S donors has shown that only GYY4137 (1 mg/kg) inhibited 5-HT-induced hyperreactivity, and both GYY4137 and NaHS (1 mg/kg) prevented the neutrophil increase in BAL fluid in LPS-induced airway inflammation. IL-1β increase in BAL fluid was abolished by both GYY4137 and NaHS treatments whereas TNF-α levels remained unchanged. Furthermore, GYY4137 treatment did not have any effect in LPS-induced changes of lung pathology whereas NaHS prevented the paranchymal inflammation. The different H2S releasing pattern of these donors may explain the difference of their effects in this model. Compounds that provide stable H2S levels via local application may be a new therapeutic approach in airway inflammation.

Lactobacillus rhamnosusGG reduces hepatic fibrosis in a model of chronic liver disease in rats

The intestinal dysbiosis is common in chronic liver disease and can induce to inflammatory responses and mediate the collagen deposition in the liver. To evaluate the probiotic Lactobacillus rhamnosus GG (LGG) for the treatment of liver fibrosis in a model of chronic cholestatic liver disease in rats. Male adult Wistar rats (n = 29) were submitted to common bile duct ligation (BDL groups) or manipulation of common bile duct without ligation (Ctrl groups).Two weeks after surgery, each group was randomly divided to receive 1 ml of PBS (Ctrl and BDL) or PBS containing 2.5 x 107CFU of LGG (Ctrl-P and BDL-P) through gavages for 14 days. Euthanasia occurred 33 days after surgery when samples of blood and liver tissue were collected. The hepatic gene expression of Tlr4, Tnfα, IL-6, Tgfβ, and metalloproteinase-2 and -9 were higher in the BDL groups in comparison to Ctrl. The ductular reaction evaluated by immunocontent of cytokeratin-7 (CK7) and the content of collagen were increased in BDL groups. Also, there was an imbalance in the antioxidant defenses (superoxide dismutase and catalase) and an increase in the oxidative stress marker sulfhydryl in BDL groups. The treatment with LGG significantly reduced gene expression of IL-6, collagen deposition, and ductular reaction in hepatic tissue of animals from BDL-P groups. The treatment with the probiotic LGG was able to reduce liver fibrosis, ductular reaction, and hepatic gene expression of IL-6 in a model of cholestatic liver disease in rats.

Response by Luo et al to Letter Regarding Article, “Fabrication of Synthetic Mesenchymal Stem Cells for the Treatment of Acute Myocardial Infarction in Mice”

We thank Takov et al for their interest in our study.1 In their letter to the editor, Takov et al raise 3 concerns regarding our study. First, Takov et al pointed out the necessity of including a vehicle control and the importance of administration technique in the in vivo mouse myocardial infarction experiments. Previous study has indicated that vehicle control and sham-operated control show similar effect on post–myocardial infarction healing.2 We completely agree with Takov et al that administration technique is important in cell transplantation. In our study, we intramyocardially inject 1×105 synthetic mesenchymal stem cell (synMSC) particles or mesenchymal stem cells (MSCs) in 50 μL PBS into 4 sites in the border zone …

Maintenance of the contractile phenotype in corpus cavernosum smooth muscle cells by Myocardin gene therapy ameliorates erectile dysfunction in bilateral cavernous nerve injury rats.

The pathophysiology of erectile dysfunction post radical prostatectomy is not clearly clarified, and the low efficacy of traditional PDE5i treatment remains a major complaint in contemporary practice. This study aimed to demonstrate phenotypic modulation in bilateral cavernous nerve injury (BCNI) rats within 7days, and subsequently validate gene therapy with Myocardin (Mycod) by maintaining a contractile phenotype in corpus cavernosum smooth muscle cells. Initially, 36 male rats were randomly divided into BCNI and negative control (NC) groups for histological and phenotypic molecular measurements at 3, 5, and 7days. Afterwards, an additional 30 rats received a single intra-cavernous injection of 50μL PBS, Ad-Myocd (1×1011 pfu/ml) or Ad-vector for 10 animals each, namely the NC+PBS, BCNI+Ad-Myocd, and BCNI+Ad-vector groups. Finally, the validity and mechanism of Myocd transfection was explored at 21days invivo and 48h invitro. Western blotting showed canonical declines in Myocd, α-SMA, and Calponin expression, as well as elevated Osteopontin (OPN) expression, before corporeal morphological and SM-to-collagen ratio changes at day 5 after injury. Overexpression of Myocd maintained the contractile phenotype of corpus cavernosum smooth muscle cells, ameliorated bilateral cavernous nerve injury rat erectile dysfunction, as well as promoted cell contractility and suppressed proliferative capacity. Simultaneously, confocal imaging revealed up-regulation and co-localization of serum response factor in gene-transferred cells. In conclusion, our study is the first to investigate corpus cavernosum smooth muscle cells phenotypes in the early stages of cavernous injury model rats, and Myocd reversed phenotypic modulation by activating serum response factor. The experimental results demonstrated the validity of gene therapy for erectile dysfunction.

Efficacy of a repeat dose injection of CLR 131 (I-131-CLR1404) in U87-MG tumor-bearing athymic nude mice.

e14089 Background: CLR 131 is a novel radioiodinated therapeutic that exploits the selective uptake and retention of phospholipid ethers (PLEs) by malignant cells. Study was to evaluate the therapeutic effect of CLR 131 when administered as a repeat dose intravenous injection in U87-MG tumor bearing mice. Malignant gliomas are associated with a low survival rate, and treatment with chemotherapy, external beam radiation, or surgery have shown limited effectiveness. In this study, therapeutic effect was evaluated by measurement of tumor volume increase and by survival of CLR 131 treated animals compared to animals dosed with a I-127-CLR1404 control. Methods: The U87-MG cell line (human glioma) was purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in MEM media supplemented with 10% fetal bovine serum. Twenty female athymic nude mice (Harlan, Indianapolis, IN) weighing between 16-20 g were anaesthetized with isofluorene and inoculated subcutaneously in the right flank with 1 x 106 U87-MG tumor cells suspended in 50 μL PBS. The mice were given potassium iodide at a concentration of 0.1% in their drinking water to block possible free iodide in the drug formulation, with the addition of 0.4% (w/v) sweetener (Sweet and Low) to aid palatability, three days prior to injection and continuing through two weeks post injection. Doses of 95.7µCi and 109.0 µCi of CLR 131were given at Day 0 and Day 7 (N = 8 per group). Control doses of I-127-CLR1404 were given at the indicated dose and was injected via tail vein on Day 0 and Day 7. Results: The control group’s tumors increased 25-fold over a period of four weeks and the treatment group’s tumors increased 12-fold over the same period. There was a significant difference between the control and treatment tumor volumes. Mean survival time was 24.5 days and 31.9 days post injection, respectively. Conclusions: CLR 131 represents a unique, first in class targeted radiotherapeutic currently in clinical development. This study shows that CLR 131 dosed twice provides significant inhibition of tumor growth in a U87-MG tumor model which is known for very aggressive growth. Additionally, CLR 131 provided an overall survival benefit over the control group.

α-Galactosylceramide and all trans-retinoic acid work together to promote antigen presentation and antibody production

Abstract α-galactosylceramide (αGalCer) and all trans-retinoic acid (RA) are both known as potent immune regulators on various steps of the immune responses. Our previous studies have shown that αGalCer and RA could regulate B cell differentiation in vitro and increase antibody production in vivo. To investigate the underlying mechanisms related to the antibody response, animal experiments (in 8-wk old female Balb/c mice) were performed to determine the antigen presentation and B cell response during primary immunization. In a one-day experiment, dextran-FITC or KLH-FITC was injected intravenously with or without αGalCer (2 μg in 100 μl PBS with antigen, iv) or RA (37.5 μg in 10 μl canola oil, oral dose); in a 10-day experiment, KLH was injected (100 μg/100 μl PBS) with or without αGalCer/RA treatment. Spleens were analyzed by flow cytometry and immunofluorescent staining. We observed that Dextran and KLH after 24h of injection were localized at the spleen marginal zone (MZ) area, with only KLH colocalized with the metallophilic macrophages; in the presence of αGalCer and RA, the band of metallophilic macrophages became wider. While dextran moved markedly toward the follicular area after treatment, KLH signal was not seen within the follicle, and also became weaker at the MZ area, suggesting the different presentation and processing mechanisms of the antigens. At day 10, substantially more active germinal centers were formed in the presence of αGalCer and RA, with more focused staining of CD23+GL-7+ cells, markers of germinal center B cells. B cell numbers were increased at both time points. Our study suggests that αGalCer and RA together may promote antigen presentation and induce B cell differentiation that ultimately increases antibody production.

Effects Of Acute Eccentric Exercise On Primary Antibody Responses To Ovalbumin Vaccination In Aged Mice.

Several studies have suggested that acute eccentric exercise can enhance vaccination responses in humans. However, the underlying mechanisms have remained understudied. We have shown that acute eccentric exercise does not improve the antibody responses to ovalbumin (OVA) vaccination in young mice. Thus, we replicated the study in an immunosenescence animal model, where there is reduced vaccine efficacy. PURPOSE: To determine the effects of acute eccentric exercise on the primary antibody response to vaccination in aged mice. METHODS: C57BL/6 male mice, aged 27 months (n=16) were randomized into either eccentric exercise (ECC, n=8) or sedentary (SED, n=8) groups. For the ECC group, mice were exercised at 17m/min at -20% grade for 45 min on a treadmill. SED mice remained in their home cages. All mice were inoculated in the gastrocnemius with 25 μg of OVA and 200 μg aluminum hydroxide (a suboptimal dosage based on titration experiments) in 50 μl sterile saline immediately after the exercise. Blood was collected prior to, and one, two and four weeks after vaccination. ELISA was performed to analyze anti-OVA IgG. At three weeks post exercise, all mice were injected with 100 μg OVA dissolved in 10 μl PBS into the dorsal side of the right ear to determine the delayed-type hypersensitivity (DTH) response. The left ear received 10 μl PBS alone as a control. Ear thickness was measured immediately before, 24 h and 48 h after intradermal injection. The measurements were performed in triplicate, where researchers were blinded to treatment. Maximum ear swelling occurred at 24 hours post injection. Results were expressed as the difference between the right and left ear thickness. RESULTS: We found a significant time main effect (p<0.001) indicating a significant increase in anti-OVA IgG at one, two and four weeks relative to pre-immunization. However, there were no significant time x treatment (p=0.652) nor treatment main effects (p=0.764). There was a significant difference between ECC and SED groups in ear DTH at 24 h post injection (p = 0.028), indicating eccentric exercise increased the DTH response. CONCLUSION: Acute eccentric exercise immediately before vaccination improved the DTH (i.e. cell-mediated immune), but not the antibody, response to vaccination in aged mice.

Effects of intrauterine infusion of Escherichia coli lipopolysaccharide on uterine health, resolution of purulent vaginal discharge, and reproductive performance of lactating dairy cows

The objectives of the current experiment were to evaluate the effects of intrauterine infusion of Escherichia coli lipopolysaccharide (LPS) in cows diagnosed with purulent vaginal discharge (PVD) on intrauterine cell population, resolution of PVD, uterine health, and reproductive performance. Jersey cows (n = 3,084) were examined using the Metricheck device to diagnose PVD at 35 ± 6 d postpartum. Purulent vaginal discharge was defined as the presence of purulent (≥50% pus) discharge detectable in the vagina. Of the 310 cows positive for PVD, 267 cows were enrolled in the current experiment. To ensure proper timing of treatment and collection of samples, only 9 PVD-positive cows were treated per day. Selected cows were balanced at 35 ± 6 d postpartum for lactation number, body condition score, and milk yield and were randomly assigned to receive an intrauterine infusion of 20 mL of phosphate-buffered saline (PBS; control, n = 87), 20 mL of PBS with 150 µg LPS (LPS150, n = 91), or 20 mL of PBS with 300 µg of LPS (LPS300, n = 89). Uterine cytology was performed immediately before treatment and 1, 2, and 7 d after treatment to evaluate the effect of LPS treatment on intrauterine cell population. Cows were examined with the Metricheck device at 7 and 28 d after treatment to evaluate the effects of treatment on resolution of PVD. Reproductive status was recorded up to 200 d postpartum. Cows diagnosed with PVD had greater incidence of twinning, dystocia, retained placenta, and metritis after calving than cows without PVD. Count of polymorphonuclear leukocytes (PMNL) in uterine cytology 1, 2, and 7 d after intrauterine infusion was not statistically different among treatments. From d 0 to 1, however, PMNL count in uterine cytology of PBS cows increased by 5%, whereas the PMNL count in uterine cytology of LPS150 and LPS300 cows increased by 54 and 48%, respectively. Treatment did not affect the likelihood of cows being diagnosed with PVD 7 and 28 d after intrauterine infusion. Cows without PVD and LPS150 cows were more likely to be pregnant after the first postpartum AI than PBS cows. After the second postpartum AI, cows without PVD were more likely to be pregnant than PBS and LPS300 cows. Hazard of pregnancy up to 200 d postpartum was decreased for PBS and LPS300 cows compared with cows without PVD, and it tended to be decreased for LPS150 cows compared with cows without PVD. Intrauterine treatment with 150 µg of E. coli LPS of cows diagnosed with PVD improved likelihood of pregnancy after the first postpartum AI, but further research is needed to elucidate the mechanism by which LPS treatment improved fertility.

Expression changes of transforming growth factor-β1 and fibronectin in bovine trabecular meshwork cells cultured by iris pigment particles

Background Pigmentary glaucoma and pseudoexfoliation glaucoma are characterized by pigment dispersion in trabecular meshwork, and the dipersitional pigment probably contributes to the resistance of aqueous outflow pathway, irreversible damage of the trabecular meshwork and the remodeling abnormality of extracellular matrix.Researches determined that contents of transforming growth factor-β (TGF-β) in the aqueous humor are increased in glaucomatous eyes.However, the effects of TGF-β and fibronectin (FN) in the trabecular meshwork cells (TMCs) acted by iris pigmentary particles still are not elucidated. Objective This study was to investigate the effects of iris pigment particles on TGF-β1 and FN expression in bovine TMCs (BTMCs) cultured in vitro. Methods BTMCs were cultured in vitro by explant culture method and identified by morphological evaluation.The third generation of BTMCs were divided into normal control group and pigment group, and 100 μl PBS and 100 μl iris pigment suspension (final concentration of 1×107 particles/ml) were added into the medium for 24 hours, respectively.The expressions of TGF-β1 mRNA, FN mRNA and their proteins in the BTMCs were assayed by real-time fluorescence quantitative PCR and ELISA, respectively. Results Cultured cells grew well and showed the fusiform, polygon and dendritic like in shape, with pigmented and round nuclei.The relative expression levels of TGF-β1 mRNA and FN mRNA in the cells were 2.98±0.27 and 0.36±0.10 in the iris pigment group, which were significantly higher than 1.00±0.00 and 1.00±0.00 in the normal control group (t=12.68, 10.60, both at P=0.00). The concentrations of TGF-β1 protein and FN protein in the cell suspension were (156.60±9.74)ng/L and (59.29±15.79)mg/ml in the iris pigment group, showing significant differences in comparison with (65.46±14.24)ng/L and (102.10±12.14)mg/ml in the normal control group (t=9.15, P=0.00; t=3.72, P=0.02). Conclusions The expression of TGF-β1 is up-regulated and that of FN is down-regulated in BTMCs cultured by iris pigment, inferring that TGF-β1 and FN participate in the pathogenesis and development of pigmentary and pseudoexfoliation glaucoma. Key words: Glaucoma; Gene expression regulation; Iris/metabolism; Melanins/metabolism; Trabecular meshwork/metabolism; Transforming growth factor beta1/metabolism; Fibronectin; Cells, cultured

HP-01-005 Effectiveness of intracavernous delivery of recombinant human hepatocyte growth factor on erectile function in the streptozotocin-induced diabetic mouse

Diabetic erectile dysfunction (ED) is a disease mostly of vascular origin with severe endothelial dysfunction and responds poorly to oral phosphodiesterase-5 inhibitors. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and angiogenesis. Here, we determined the effectiveness of recombinant human hepatocyte growth factor (rh-HGF) protein in restoring erectile function in diabetic mice. Intracavernous injections of phosphate-buffered saline (days -3 and 0; 20 μL), a single intracavernous injection of rh-HGF protein (day 0; 4.2 μg/20 μL of PBS), or two successive intracavernous injections of rh-HGF protein (days -3 and 0; 4.2 μg/20 μL, respectively). Two weeks after the treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic and biochemical studies. Repeated intracavernous injections of rh-HGF protein induced significant restoration of erectile function in diabetic mice (90% of control values), whereas a single intracavernous injection of rh-HGF protein elicited modest improvement. Rh-HGF significantly increased the content of endothelial cells, pericytes, and smooth muscle cells and decreased the generation of reactive oxygen species (superoxide anion and peroxynitrite) and extravasation of oxidized LDL in diabetic mice. Rh-HGF protein promoted proliferation and decreased apoptosis in primary cultured mouse cavernous endothelial cells and pericytes in vitro. Under high-glucose condition, rh-HGF protein also promoted tube formation in MCECs and enhanced neurite sprouting in MPG culture in vitro.

Isolation and Number of Gonadal Primordial Germ Cells (Gonadal PGCs) on the Stages of Early Embryonic Development of KUB Chicken

Primordial germ cells (PGCs) are cells that will differentiate themselves into spermatogonia in the testis or oogonia in the ovary. Primordial germ cells arise from epiblast and circulate through the bloodstream and finally entering gonadal anlage. The aim of this study was to determine the number of gonadal PGCs of KUB chicken at different development stages. Sixty KUB chicken fertile eggs were divided into four groups (6, 7, 8, and 9 days incubation periods), and incubated at 38 oC with a humidity of 60%. Harvesting was synchronized to the embryonic development at 6-9 d. Gonads were collected using sharp tweezers, and were placed in Eppendorf tube 1.5 mL containing 500 µL PBS [-]. Gonadal PGCs were purified using PBS [-]. The results showed that the average number of gonadal PGCs at 6, 7, 8, and 9 d were 113.7; 143.5; 92.9; and 85.7 cells per embryo, respectively. Number of gonadal PGCs per embryo of KUB chicken were significantly affected by stage of embryonic development (P<0.05), which reached a peak at day 7 of incubation, so that the isolation and collection of PGCs from the gonads were recommended at day 7 of incubation. This information is useful in production of germline chimera of other Indonesian local chickens.