Coinjection Group Research Articles

Mouse model of Graves' orbitopathy induced by the immunization with TSHR A and IGF-1R α subunit gene.

The study aimed to establish a mouse model of Graves' disease (GD) with Graves' orbitopathy (GO; GD + GO) that can represent the clinical disease characteristics. A eukaryotic expression plasmid of insulin-like growth factor 1 receptor (IGF-1R) α subunit (pcDNA3.1/IGF-1Rα) and a thyrotropin receptor (TSHR) A subunit plasmid (pcDNA3.1/TSHR-289) were injected in female BALB/c mice followed by immediate electroporation to induce a GD + GO model. Grouping was performed according to the frequency of injection (2- to 4-week intervals) and type of injected plasmids: T: pcDNA3.1/TSHR-289( +), I: pcDNA3.1/IGF-1Rα( +), or co-injection T + I: pcDNA3.1/TSHR-289( +) and pcDNA3.1/IGF-1Rα( +). Serum TSH, T4, TSAb, TSBAb,body weight, and blood glucose levels were evaluated. Thyroid 99mTcO4- imaging and retrobulbar magnetic resonance imaging (MRI) were performed, and bilateral eye muscle volumes were measured. Immunohistochemistry and hematoxylin-eosin staining were performed on the relevant tissues, and semi-quantitative analysis was performed. A total of 60% of mice (3/5, one mouse died) in the T group developed GD + GO. In the T + I group, 83.3% of mice (5/6) developed GD + GO. Mice in the I group did not develop GD. Compared with the control group, serum T4, TSAb, and TSBAb of the mice in the GD + GO model groups were increased to varying degrees (P < 0.05), and serum TSH and body weight were significantly lower compared to the control group (P < 0.05). The thyroid uptake capacity of 99mTcO4- and the volume of eye muscle of mice in the GD + GO group were significantly higher compared to the control group (P < 0.05). The thyroid and retrobulbar muscles of these mice showed varying inflammatory infiltration and interstitial muscle edema. The severity of GD + GO in the co-injection group was not related to injection frequency; however, GD and ocular signs in co-injection mice were more severe compared to the T group. We successfully induced a GD + GO mouse model by a repeated co-injection of pcDNA3.1/IGF-1Rα and pcDNA3.1/TSHR-289 plasmids. Injection of pcDNA3.1/IGF-1Rα alone failed to induce GD. Co-injection of two plasmids induced more severe GD + GO than pcDNA3.1/TSHR-289( +) alone.

No Difference Between One-shot Co-injection of Corticosteroids and Hyaluronic Acid in a Three-injection Regimen for Knee Osteoarthritis: A Randomized, Double-blind Trial [RETRACTED

Although many guidelines now recommend against hyaluronic acid (HA) injections alone for the nonoperative management of knee osteoarthritis (OA), prior studies have disagreed about whether adding an intra-articular corticosteroid to HA results in sufficient benefit to justify the potential dose-dependent and time-dependent chondrotoxicity of corticosteroids. (1) Do patients with knee OA treated with low-dose intra-articular corticosteroids and HA administered under ultrasound guidance have more favorable patient-reported knee OA-related outcomes from 1 to 26 weeks after treatment than those treated with ultrasound-guided HA injections alone? (2) Do patients treated with a corticosteroid co-injection exhibit more favorable physical functional performance than those treated with injections of HA alone? We conducted a randomized, double-blind trial at a small medical center in urban Taipei, Taiwan. We considered those who fulfilled the clinical and radiographic criteria for knee OA, defined by the American College of Rheumatology as a Kellgren-Lawrence grade 2 or 3, as potentially eligible. We excluded those who had a previous knee fracture or surgery and those who had received intra-articular knee injections of HA or corticosteroids within 6 months of the trial. Based on these criteria, 78 patients were eligible, and 76 agreed to be randomized to the co-injection or HA-only groups through block randomization. The groups had 38 patients each; all participants who completed the study follow-up were analyzed in the groups to which they were randomized and treated. There was no crossover. The patients in the co-injection group were treated with a one-shot co-injection of HA with corticosteroids (Hyruan Plus: molecular weight, 3000 kDa, 10 mg/mL HA, 2 mL with triamcinolone acetonide 10 mg/dL, 1 mL) in the first week followed by two injections of HA (10 mg/mL, 2 mL) at a 1-week interval. The patients in the HA-only group received three HA injections (Hyruan Plus: molecular weight, 3000 kDa, 10 mg/mL HA, 2 mL) administered at a 1-week interval. A senior physiatrist performed ultrasound-guided knee injections using the lateral suprapatellar approach. The physiatrist, participants, and outcome assessor were all blinded to the injection contents. No differences were observed between the two groups regarding primary demographic data, baseline WOMAC and Knee Injury and Osteoarthritis Outcome Score (KOOS) scores, and physical functional performance. Three participants experienced localized swelling after the injections, and all symptoms improved after a few days of localized ice pack application. A total of 3.9% of patients (three of 76) had adverse events (mainly localized swelling of the knee, with or without accompanying pain), with no between-group differences in the frequency of adverse events. No severe side effects were reported during or after the injections. The patient-reported outcomes were the WOMAC and KOOS scores. The objective outcome was physical function performance (speed for 10-meter regular and fastest walking, and time for stair-climbing and chair-rising). The primary outcome timepoint was 6 months postinjection. A sample size of 34 participants was required in each group, based on a difference in WOMAC scores of 58 points, a significance level of 0.05, and a power of 0.8. We found no difference in improvement from baseline between the two groups regarding patient-reported outcomes at any follow-up timepoint, from 1 week after injection to 6 months. At 6 months post-treatment, compared with baseline, there were no between-group differences in improvement between the co-injection group and the HA-only group in terms of WOMAC pain scores (mean difference 1 [95% CI -2 to 3]; p = 0.62), stiffness (mean difference -11 [95% CI -13 to -9]; p < 0.001), and physical function (mean difference -4 [95% CI -5 to -3]; p < 0.001) and KOOS scores for pain, other symptoms, activities of daily living, sports and recreation, and quality of life. Except for chair-rising time at 6 months, which may have represented spurious significance because of the number of comparisons, compared with baseline, we found no differences in improvement between the two groups regarding the regular and fastest walking speed and stair ascent and descent time. We found that a single co-injection of corticosteroids along with HA did not confer any additional benefit regarding patient-reported outcomes or physical function compared with a three-injection regimen of HA. Therefore, we do not recommend a one-shot co-injection of corticosteroids alongside the three-injection regimen of HA for patients with knee OA, and because prior research found HA to be ineffective, HA should not be used with or without corticosteroids for patients with knee OA. Level I, therapeutic study.

Revisiting the “Danger Theory”: Toll-like Receptor 9 Stimulation Triggers Activation of Conventional CD8α+ and Plasmacytoid Dendritic Cells En Route to Enhancing FVIII Inhibitor Formation

Inhibitor formation is the most serious complication of FVIII replacement therapy for hemophilia A. The long-standing "danger theory" posits that inhibitors may form as part of collateral damage from immune response to a primary challenge such as an infection or vaccination. Innate immune signaling, for example through Toll-like receptors (TLRs), could be one way of triggering or reinforcing unwanted immune responses. However, the danger hypothesis has been contradicted by recent reports showing no increase in inhibitor formation in boys and animal models with hemophilia when FVIII was administered concurrently with vaccines. The aim of this study was to elucidate the influence of TLR9 signaling on FVIII inhibitor formation in hemophilia A mice. Hemophilia A (F8e16-/-) B6/129 mice were co-injected IV with FVIII (1.5 IU) and ODN-1826 (a class B CpG oligodeoxynucleotide, 50 µg), which is a TLR9 agonist. Control mice were naïve or received FVIII only. Blood samples and spleens were collected for Bethesda assay and flow cytometry analysis 3h, 24h, 2, 3 and 7 days or 2, 4, 6 and 8 weeks after a single or repeated once-weekly injections. After four weeks, mice co-injected with FVIII and ODN-1826 (n=4) showed ~15-fold higher inhibitor titers (median 2667 BU/mL) than mice injected with FVIII only (median 181.4 BU/mL; n=15). We also found significantly higher T follicular helper (Tfh; CD4+CXCR5+PD1+Bcl6―) [F (7, 96) = 9.801, p&amp;lt;0.0001] and Germinal Center (GC) B cell numbers (CD19+GL7+CD95+) [F (6, 92) = 11,53, p&amp;lt;0.0001] in the spleen after 4, 6 and 8 weeks of co-injections, with the Tfh numbers being 1.3-, 12.2- and 1.65-fold, while GC B cell numbers being 2.9-, 5.3- and 1.1-fold higher, respectively, compared to mice injected with FVIII only. GC B cell numbers correlated with inhibitor titers [r(31)=0.52, p=0.002]. We also investigated dendritic cell (DC) responses after a single injection of FVIII or co-injection with ODN-1826. We found increased numbers of monocyte-derived DCs (moDCs; CD11chighMHCII+CD11b+ CD64+MAR-1+) in the co-injected group at all time points (3, 24, 48, 72 hours and 1 week after a single injection; n=4-5 per group) with the peak number (~10-fold higher compared to naïve mice; p=0.0002) being reached 72 hours and remaining similarly elevated at 1 week. However, levels of activation markers CD86 and MHCII on moDCs in both injected groups were not significantly elevated. The moDC numbers were also ~2.5-fold higher in the FVIII only group than in naïve animals at one week post-administration, but the difference was not statistically significant. Conversely, exposure to FVIII with or without ODN-1826 did not significantly affect the number of CD8α+CD11b- DCs, but this subset showed &amp;gt;2-fold upregulation of both CD86 and MHCII in the co-injected group 3h and 24h after injection (n=5-11, p&amp;lt;0.01). We further found increased numbers of plasmacytoid DCs (pDCs; CD11c+CD11b―PDCA+) but the difference was significant only in the co-injected group at d1 and d7 after injection (~2-fold higher than the naïve group; n=4-16 per group, p&amp;lt;0.05). This DC subset also showed upregulated CD86 and MHCII levels ~2-fold in the co-injected group, with difference between CD86 levels showing significance (p=0.0024). We next examined an interferon signature in pDCs by intracellular cytokine staining and found that the cells from co-injected mice had ~2-fold higher levels of IFNβ (p=0.0001, n=5 per group). We propose that TLR9 stimulation enhances FVIII inhibitor formation in hemophilia A mice through recruitment and/or activation of conventional CD8α+ and plasmacytoid dendritic cells. The narrowing differences in Tfh and GC B cell numbers between the FVIII only and ODN-1826 co-injected groups at week 8 suggest that TLR9 stimulation accelerates GC formation in response to FVIII, which otherwise may reach a similar magnitude, but it takes longer. Also, our results suggest that it may be premature to put the danger theory to rest. The outcome of an immune challenge may vary depending on which innate immune receptor is concurrently stimulated. Notably, all vaccinations tested in the animal study showing no link to inhibitor formation were against pathogens with genomes composed of single-stranded RNA (measles, mumps, rubella and influenza viruses), which does not activate TLR9. Therefore, the impact of vaccination against single-stranded DNA pathogens that can stimulate TLR9, such as VZV, may be worth further investigation. Disclosures Herzog: Takeda Pharmaceuticals: Patents &amp; Royalties.

Glycyl-l-glutamine attenuates NPY-induced hyperphagia via the melanocortin system

This study aimed to determine whether glycyl-l-glutamine (Gly-Gln; β-endorphin (30–31)), a non-opioid peptide derived from β-endorphin processing, modulates neuropeptide Y (NPY)-induced feeding and hypothalamic mRNA expression of peptide hormones in male broiler chicks. Intracerebroventricular injection of NPY (235 pmol) generated a hyperphagic response in ad libitum chicks within 30 min. Co-administration of Gly-Gln (100 nmol) attenuated this response, inducing a 30 % decrease. This was not attributable to Gly-Gln hydrolysis because co-administration of glycine (Gly) and glutamine (Gln) had no effect on NPY-induced hyperphagia. Gly-Gln injected alone also showed no effect. The hypothalamic pro-opiomelanocortin mRNA expression in the co-injection group was significantly higher than that in the NPY alone group. These data indicate that endogenous Gly-Gln may contribute to regulate feeding behavior via the central melanocortin system in chicks and acts as a counter regulator of the neural activity in energy metabolism.

Randomized, Double-Blinded, Sham-Controlled, Split-Hand Trial Evaluating the Safety and Efficacy of Triamcinolone Acetate Injection After Calcium Hydroxylapatite Volume Restoration of the Dorsal Hand.

Calcium hydroxylapatite (CaHA) is currently the only FDA-approved soft-tissue filler indicated for augmentation of the dorsal hand. Although the treatment is generally safe and effective, adverse side effects such as swelling and edema postinjection are common and can sometimes be debilitating. In this study, the authors explore the utility of triamcinolone acetate coinjection with CaHA to the dorsal hands to mitigate adverse effects and improve patient experience. After obtaining informed consent, 20 subjects were enrolled in this double-blind, randomized, split-hand, sham-controlled clinical trial. Subject hands were randomized to receive either CaHA with triamcinolone acetate or CaHA with sham saline coinjection. Follow-up evaluations were performed by a blinded investigator at Days 7, 14, 30, 90, 180, 270, and 360 and consisted of the validated Merz Hand Grading Scale and assessments of erythema, edema, modules, bruising, and skin atrophy. Subjects also kept a daily diary for the first 30 days postinjection documenting bruising, itching, pain, redness, swelling, difficulty in performing activities with hands, and sensory alteration in hands. There were no significant differences in treatment efficacy between the 2 groups. Post-treatment swelling was significantly reduced between Days 6 and 19 in the triamcinolone acetate coinjection group. The addition of triamcinolone acetate coinjection with CaHA for dorsal hand augmentation did not negatively impact treatment efficacy but significantly reduced adverse side effects. This strategy represents a safe and effective way to improve patient experience and treatment tolerability.

C-Kit-Positive Adipose Tissue-Derived Mesenchymal Stem Cells Promote the Growth and Angiogenesis of Breast Cancer

Background Adipose tissue-derived mesenchymal stem cells (ASCs) improve the regenerative ability and retention of fat grafts for breast reconstruction in cancer patients following mastectomy. However, ASCs have also been shown to promote breast cancer cell growth and metastasis. For the safety of ASC application, we aimed to identify specific markers for the subpopulation of ASCs that enhance the growth of breast cancer. Methods ASCs and bone marrow-derived vascular endothelial progenitor cells (EPCs) were isolated from Balb/c mice. c-Kit-positive (c-Kit+) or c-Kit-negative (c-Kit−) ASCs were cocultured with 4T1 breast cancer cells. Orthotropic murine models of 4T1, EPCs + 4T1, and c-Kit+/-ASCs + 4T1/EPCs were established in Balb/c mice. Results In coculture, c-Kit+ ASCs enhanced the viability and proliferation of 4T1 cells and stimulated c-Kit expression and interleukin-3 (IL-3) release. In mouse models, c-Kit+ASCs + 4T1/EPCs coinjection increased the tumor volume and vessel formation. Moreover, IL-3, stromal cell-derived factor-1, and vascular endothelial growth factor A in the c-Kit+ASCs + 4T1/EPCs coinjection group were higher than those in the 4T1, EPCs + 4T1, and c-Kit−ASCs + 4T1/EPCs groups. Conclusions c-Kit+ ASCs may promote breast cancer growth and angiogenesis by a synergistic effect of c-Kit and IL-3. Our findings suggest that c-Kit+ subpopulations of ASCs should be eliminated in fat grafts for breast reconstruction of cancer patients following mastectomy.

In vivo protective effect of phosphatidylcholine on carbon tetrachloride induced nephrotoxicity

Phosphatidylcholine (PC) from egg yolk is a bioactive substance with various beneficial effects, including anti-inflammatory and anti-oxidant effects. Recently, this substance has been reported to prevent acute hepatotoxicity. In the present study, we aimed to evaluate the putative protective effect of PC on carbon tetrachloride (CCl4)-induced nephrotoxicity in ICR mice. Many previous studies demonstrated that CCl4 induces nephrotoxicity resulting in renal oxidative damage. CCl4 in corn oil (0.1ml, 1.2g/kg) was intra-peritoneally injected into 7-week-old ICR mice twice a week. PC in corn oil (0.1ml, 100mg/kg) was then orally injected daily for a week. In 7 days, blood urea nitrogen (BUN) and creatinine concentrations had significantly increased in the CCl4 group compared to the control group, whereas the PC and CCl4 co-injected group had significantly decreased BUN and creatinine concentrations compared to the CCl4 group. Comparative analysis of histopathological injuries revealed that PC abrogated the nephrotoxicity of CCl4 at 7 days. Accordingly, PC also improved renal fibrosis induced by CCl4. Various biomarkers associated with oxidative damage appeared to be up-regulated in the CCl4 group, whereas in the PC and CCl4 co-injected group, levels of oxidative damage significantly decreased. Aquaporin1 (AQP1), an important water transport protein in the kidney, was down regulated in the CCl4 group compared to the control group. PC counteracted this effect. These results strongly suggest that PC can protect against oxidative damage induced by CCl4 in the kidney and enhance recovery from renal disorders.

Effects of artificial infection of Litopenaeus vannamei by Micrococcus lysodeikticus and WSSV on the activity of immunity related enzymes.

In this study, the activities of 5 immunity related enzymes namely acid phosphatase (ACP), alkaline phosphatase (AKP), phenoloxidase (PO), peroxidase (POD) and lysozyme phosphatase (LZM)) of Litopenaeus vannamei after they have been injected with different concentrations of Micrococcus lysodeikticus and the white spot syndrome virus (WSSV) were examined. The cumulative mortality at 0, 24, 48, 72, 96 h was obtained. Copy numbers of WSSV in L. vannamei after a single infection, secondary infection and concurrent infection were measured. Hemolymph samples of M. lysodeikticus and WSSV injected shrimp were collected at 0, 6, 12 24, 48, 72, 78, 84, 96 and 120 h. The results were: (i) The cumulative mortality of L. vannamei increased as the shrimp were infected with higher concentration of the bacteria; (ii) The most sensitive changes of ACP, AKP and LZM were in the 6.2 × 10(5), 6.2 × 10(6), 6.2 × 10(7) cfu/mL M. lysodeikticus group; (iii) ACP but LZM were more sensitive to M. lysodeikticus than WSSV, and AKP, PO and POD is more sensitive to WSSV; (iv) The copies of WSSV in the co-injected group were higher than WSSV-single infection and WSSV-bacteria-secondary infection group at 48 h. The amount of WSSV in L. vannamei of concurrent infection and WSSV-bacteria-secondary infection groups were higher than that of the WSSV-single infection group.

Differential And Cooperative Roles Of β‐Catenin And Yap During Hepatoblastoma Pathogenesis

Hepatoblastoma (HB) is the most frequent pediatric liver tumor. In our previous study we demonstrated that coordinated activation of Yap and β‐catenin is a major mechanism in promoting HB development. However, the individual contribution of Yap and β‐catenin and how they may interact to contribute to induce HB remains unknown. Here, instead of co‐delivering yap and β‐catenin sleeping beauty plasmids with transposase, we hydrodynamically delivered either Yap first followed 3 days or 7 days later by β‐catenin or vice versa. The mouse livers were harvested at 6 weeks, 7 weeks, and 9 weeks after the second injection for comparing tumor size and phenotype to the Yap‐b‐catenin co‐injected group. There was no difference in the liver phenotype when Yap injection preceded β‐catenin compared to Yap‐β‐catenin co‐injection. However, the tumors were significantly smaller and fewer when β‐catenin injection preceded Yap as compared to the co‐delivery group at the early time‐points (6 &amp; 7 weeks). Intriguingly, there was no difference in tumors between all 3 groups at late stage (9 weeks). Lastly, when Yap with defective TEAD binding and b‐catenin or b‐catenin with defective TCF4 binding and Yap were co‐delivered, there was alleviation of tumorigenesis. In summary, Yap is important early in reprograming hepatocyte to hepatoblasts, while both Yap and β‐catenin with preserved ability to bind their canonical transcription factors are required for tumor growth and progression through activation of downstream targets.

Synergic effects of EPI-NCSCs and OECs on the donor cells migration, the expression of neurotrophic factors, and locomotor recovery of contused spinal cord of rats.

Cell-based therapy is a promising strategy for the repair of spinal cord injury (SCI), and the synergic effects of donor cells are emphasized in recent years. In this study, epidermal neural crest stem cells (EPI-NCSCs) and olfactory ensheathing cells (OECs) were transplanted into the contused spinal cord of rats separately or jointly at 1week after injury. At 3 and 9weeks posttransplantation, migration of the donor cells, expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) and functional recovery of the contused cord were determined by techniques of histopathology, quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry and Basso-Beattie-Bresnahan (BBB) score. The results showed that the migration and distribution of EPI-NCSCs in vivo were promoted by OECs at 3weeks after transplantation, but they vanished at 9weeks. The expression of BDNF and GDNF was significantly increased by co-transplantation at molecular and protein level. Although the expression of both factors in EPI-NCSCs- and OECs-injected group was lower than in co-injected group, it was higher than in control groups. Similarly, the best locomotor recovery of the contused cord was acquired from co-injected animals. As we know, this is the first time to study the synergic effects of EPI-NCSCs and OECs, and the data indicates that donor cells migration, expression of neurotrophic factors (NTFs), and recovery of motor function can be improved by EPI-NCSCs and OECs synergistically.

Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

BackgroundFat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis.Methodology/Principal FindingsHuman MDA-MB-231 breast cancer cells represents “triple negative” breast cancer that exhibits early micrometastasis to multiple mouse organs [1]. Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells.ConclusionsHuman ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231 breast tumor xenografts to multiple mouse organs. MDA-MB-231 tumors co-injected with ASCs from one donor exhibited partial EMT, expression of MMP-9, and increased angiogenesis.

Abstract 2604: Involvement of circulating fibrocytes in the progression of adenocarcinomas by modulating EMT and tumor microenvironment.

Abstract Background: Circulating fibrocytes (CFs) are bone marrow derived, mesenchymal progenitor cells that have emerging role in many diseases. CFs was shown to participate in tissue remodeling in pulmonary hypertension and fibrosis, via secretion of different cytokines and growth factors. Nevertheless, their role in the lung cancer still has to be delineated. Thus, our aim is to identify the role of CFs in lung cancer progression and metastatic potential. Results/methods: We generated CFs by isolating human peripheral blood mononuclear cells (PBMCs) and culturing them for 10 days until they differentiated into CFs. Purity (≥95%) of the CFs population was analyzed by flow cytometry and immunofluorescence. Co-culturing of A549 (human alveolar adenocarcinoma cells) and CFs for 12 and 24 hrs, followed by collection of conditioned medium (CM) and RNA from both cells yielded interesting results. Co-cultured CM promoted proliferation (1.31±0.07 versus 1.5±0.13) and migrating capacity (64.6±26.5 versus 472.8±103.4) of A549 tumor cells. In addition, co-cultured CM caused epithelial-to-mesenchymal transition (EMT) of A549 cells; epithelial markers (E-cadherin, cytokeratin) were downregulated, and mesenchymal markers (α-smooth muscle actin, fibronectin) were upregulated. In vivo to study the CFs involvement in lung cancer, we co-injected CFs with A549 cells or A549 cells alone and measured the tumor growth after 28 days. The tumor size was significantly increased (1354.8±333.6 versus 3042.4±373.4) in co-injected group (CF+A549) compared to A549 alone. Screening for genes regulated in co-injected group tumors showed regulation of tumor microenvironment, an increase in macrophage markers (CSF1R, CD68, CD200, MMD), angiogenesis markers (EDNRB, THBS1) and ECM remodeling markers (MMP14, CTSB, CTSH) compared to A549 tumor alone. Conclusions: We believe that circulating fibrocytes may play positive role in the tumor growth and progression. The increase in EMT and migration, may suggest their involvement in invasion and metastasis. Targeting CFs and their secretory molecules can be of therapeutic importance in lung cancer. Citation Format: Alina Asafova, Vandana Nikam, Anja Schmall, Werner Seeger, Robert Voswinckel, Rajkumar Savai. Involvement of circulating fibrocytes in the progression of adenocarcinomas by modulating EMT and tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2604. doi:10.1158/1538-7445.AM2013-2604

Covalently linking poly(lactic-co-glycolic acid) nanoparticles to microbubbles before intravenous injection improves their ultrasound-targeted delivery to skeletal muscle.

Intravenously injected nanoparticles can be delivered to skeletal muscle through capillary pores created by the activation of microbubbles with ultrasound; however, strategies that utilize coinjections of free microbubbles and nanoparticles are limited by nanoparticle dilution in the bloodstream. Here, improvement in the delivery of fluorescently labeled ≈150 nm poly(lactic-co-glycolic acid) nanoparticles to skeletal muscle is attempted by covalently linking them to albumin-shelled microbubbles in a composite agent formulation. Studies are performed using an experimental model of peripheral arterial disease, wherein the right and left femoral arteries of BalbC mice are surgically ligated. Four days after arterial ligation, composite agents, coinjected microbubbles and nanoparticles, or nanoparticles alone are administered intravenously and 1 MHz pulsed ultrasound was applied to the left hindlimb. Nanoparticle delivery was assessed at 0, 1, 4, and 24 h post-treatment by fluorescence-mediated tomography. Within the coinjection group, both microbubbles and ultrasound are found to be required for nanoparticle delivery to skeletal muscle. Within the composite agent group, nanoparticle delivery is found to be enhanced 8- to 18-fold over 'no ultrasound' controls, depending on the time of measurement. A maximum of 7.2% of the initial nanoparticle dose per gram of tissue was delivered at 1 hr in the composite agent group, which was significantly greater than in the coinjection group (3.6%). It is concluded that covalently linking 150 nm-diameter poly(lactic-co-glycolic acid) nanoparticles to microbubbles before intravenous injection does improve their delivery to skeletal muscle.

Hepatitis B virus surface antigen can activate dendritic cells and modulate T helper type immune response

Hepatitis B virus surface antigen (HBsAg) is a major antigen of hepatitis B virus (HBV). Dendritic cells (DC) of HBV carriers have been reported to exhibit functional impairment. In this study, the role of HBsAg on mice bone marrow-derived dendritic cells and immune responses in vivo was studied. The immune modulatory function of HBsAg was explored by using mice bone marrow-derived dendritic cells in vitro and also by examining an ovalbumin (OVA) specific immune response in vivo. Treatment of dendritic cells with HBsAg resulted in enhanced cell surface expression of cluster of differentiation (CD) 80, CD83, CD86, and major histocompatibility complex (MHC) class II, and enhanced production of interleukin (IL)-12 p40 and IL-12 p70. Treatment of dendritic cells with HBsAg resulted in decreased T cell secretion of IL-5 by OVA stimulation. In addition, the results showed stronger OVA-specific immunoglobulin (Ig) M and weaker IgG responses in mice sera when they had been immunized with OVA and co-injected with HBsAg. It was also found that the mice exhibited significant enhancement of anti-OVA IgG2a antibody (Ab), as well as marked inhibition of IgG1 Ab production. In cellular immune responses, IL-5 production was significantly decreased and interferon (IFN)-γ increased in the group co-injected with HBsAg. On the other hand, the induction of lymphoproliferative response to OVA stimulation in spleen cells was decreased in the HBsAg co-injected group. These results demonstrate that HBsAg can affect the differentiation of T helper (Th) cells, which might provide a strategy for improving its prophylactic and therapeutic efficacy.

Adipose tissue‐derived stem cells promote prostate tumor growth

Recent evidence indicates that cancer stem cells play an important role in tumor initiation and maintenance. Additionally, the effect of tissue-resident stem cells located in the surrounding healthy tissue on tumor progression has been demonstrated. While most knowledge has been derived from studies of breast cancer cells, little is known regarding the influence of tissue resident stem cells on the tumor biology of prostate cancer. Twenty male athymic Swiss nu/nu mice (age: 6-8 weeks) were randomized into two treatment groups: (1) subcutaneous injection of 10(6) MDA PCa 118b human prostate cancer cells into the upper back or (2) subcutaneous injection of 10(6) MDA PCa 118b cells mixed directly with 10(5) GFP-labeled human adipose tissue-derived stem cells (hASCs). Tumor growth and volumes over the ensuing 3 weeks were assessed using calipers and micro-computed tomography. Immunohistochemistry was performed to identify engrafted hASCs in tumor sections. At 3 weeks after injection, the mean tumor volume in the MDA PCa 118b/hASC co-injection group (1019.95 ± 73.49 mm(3)) was significantly higher than that in the MDA PCa 118b-only group (308.70 ± 21.06 mm(3)). Engrafted hASCs exhibited the nuclear marker of proliferation Ki67 and expressed markers for endothelial differentiation, indicating their engraftment in tumor vessels. Our study revealed for the first time that ASCs subcutaneously co-injected with prostate cancer cells engraft and promote tumor progression. Further evaluation of the cross-talk between tumor and local tissue-resident stem cells may lead to new strategies for prostate cancer therapy.

Tumor uptake of 68Ga-DOTA-Tyr3-octreotate: animal PET studies of tumor flow and acute somatostatin receptor modulation in the CA20948 rat model

Factors determining the in vivo uptake of radiolabeled somatostatin analogs by neuroendocrine tumors are poorly known. The aim is to evaluate in vivo tumor perfusion and regulation of somatostatin receptors (sstr) following acute exposure to octreotide, in an animal model of neuroendocrine tumor. H(2)(15)O flow studies were performed in 8 CA20948 tumor-bearing rats and another 36 rats underwent three [(68)Ga]-DOTA-Tyr(3)-octreotate imaging sessions at 24-h intervals. After baseline (Day 0) imaging, scanning was repeated on Day 1 after octreotide injection (175 microg/kg), with a variable delay: no injection (controls, n=7), coinjection (n=6), and octreotide injection 20 min (n=7), 2 h (n=8) and 4 h (n=8) before imaging. Repeat images without octreotide was performed at Day 2 followed by sacrifice and tumor counting. H(2)(15)O studies failed to measure quantitative tumor perfusion in this model. On Day 1, ratio of tumor uptake to Day 0 was 1.2+/-0.3 in controls; 0.6+/-0.2 in the coinjection group; 0.9+/-0.2, 1.1+/-0.1 and 1.2+/-0.2 in the other groups, respectively. Uptake in the coinjection group showed a statistically significant reduction of tumor uptake (P<.0001). All groups showed increased uptake on Day 2, without statistical differences between groups. In vivo tumor counts showed good correlation with ex vivo countings (R(2)=0.946). Acute exposure to unlabeled octreotide in this neuroendocrine tumor model results in a rapid recycling or resynthesis of sstr. Positron emission tomography (PET) allowed to reliably assess quantitative uptake of [(68)Ga]-DOTA-Tyr(3)-octreotate over time in the same animal, but failed in this model to measure tumor perfusion.

Intrathecal lamotrigine blocks and reverses antinociceptive morphine tolerance in rats.

Chronic administration of morphine leads to the development of tolerance. We investigated the effects of intrathecal lamotrigine on the spinal morphine tolerance in rats that are undergoing tail flick tests. Sprague-Dawley rats were given intrathecal injections of saline 10 microl, lamotrigine 300 microg, morphine 15 microg or lamotrigine plus morphine combinations for 7 days (lamotrigine was given for days 1-7, days 1-3 or days 5-7). The acute and chronic nociceptive sensitivities were assessed using a tail flick test in which the distal 5 cm of the tail was dipped into warm water before and 30 minutes after the drug injection. With successive injections of morphine on day 8, a cumulative antinociceptive dose-response curve was constructed and the 50% effective dose (ED50) was calculated for each study group. The coinjection group of lamotrigine with morphine blocked the development of tolerance, as was shown by the preservation of morphine antinociception over 7 days and the concomitant decrease in the ED50 values on day 8, as compared with the morphine-alone group. Coinjection of lamotrigine blocked the development of morphine tolerance, as shown by the preservation of morphine antinociception over 7 days and the concomitant decrease in the ED50 values on day 8, as compared with the morphine-alone group. This study suggests that lamotrigine augments the antinociceptive action of both acute and chronic morphine therapy, and it also attenuates the antinociceptive morphine tolerance in rats.

Histological and Ultrastructural Evidence for Protective Effects on Aluminium-Induced Kidney Damage by Intraperitoneal Administration of α-Tocopherol

The nephrotoxic actions of aluminium (Al) arise from its accumulation in the kidneys, with the resultant degeneration of the renal tubular cells. It has been suggested that Al generates reactive oxygen species that cause the oxidative deterioration of cellular lipids, proteins, and DNA. To test this hypothesis, we have here investigated the potential for a protective role of alpha-tocopherol (vitamin E) during short-term exposure of rats to Al. Al was administered intraperitoneally either alone or in combination with vitamin E at a different point of abdomen, and the alterations in the kidney tissue were analyzed histologically. The results reveal that significant light microscopical and ultrastructural damage is caused by Al, whereas with the immediate coadministration of vitamin E, there is a protective effect against this damage to the kidney tissue. In Al-alone group, the glomeruli and proximal tubuli and the Bowman capsules had swellings, adherence, hemorrhage, increase in mesangial matrix, and marked interstitial tissue fibrosis, indicating severe damage. In the Al and vitamin E immediate coinjected group, renal tubule cells were almost of a normal appearance. A slight stenosis was seen in the capsular area in the Malpighi corpuscules. The tubular organization and the cytoplasmic basophilia were also much the same as in the control group, with the lumen clearly visible in most of the cortical tubuli. The results highlight the need to reduce exposure to Al, with particular attention being paid to the known sources of Al. At the same time, the maintenance of a diet that is rich in vitamin E should be beneficial in the alleviation of Al toxicity.

The Effects of Intrathecal Adenosine A1 Receptor Agonists (R-PIA) on the Morphine Tolerance in a Rat Model of Postoperative Pain

Analgesic tolerance to opioids has been described in both experimental and clinical conditions, which may limit their clinical utility. This study investigated the effects of intrathecal adenosine A1 receptor agonist (R-PIA) on spinal morphine tolerance. Methods: SD rats were given intrathecal injections of saline 10μl, R-PIA 10μg, morphine 10μg, or R-PIA plus morphine combinations for 7 days (R-PIA given for days 1-7; days 1-3; or days 5-7). Antiallodynic testing using von Frey filaments was carried out before and 30 minutes after the drug injection. On day 8, an antiallodynic dose-response curve was constructed and the 50% effective dose (ED50) for morphine (given alone) was calculated for each study group. Results: The coinjection group of R-PIA with morphine blocked the development of tolerance, as shown by the preservation of morphine antiallodynia over 7 days the concomitant decrease in the ED50 values on day 8, compared with the morphine-alone group. Although additive analgesia over days 1-7 cannot be ruled out, the reductions of the ED50 in the R-PIA and morphine combination group suggest some suppression of tolerance. Conclusions: These results suggest that intrathecal R-PIA prevents the development of spinal opioid tolerance. Future studies will be needed to examine the respective roles of supraspinal and peripheral sites of R-PIA and morphine interaction, and to investigate the mechanisms underlying the action of R-PIA on opioid tolerance.