Phosphate-buffered Saline Control Group Research Articles

LXW7 ameliorates focal cerebral ischemia injury and attenuates inflammatory responses in activated microglia in rats.

Inflammation plays a pivotal role in ischemic stroke, when activated microglia release excessive pro-inflammatory mediators. The inhibition of integrin αvβ3 improves outcomes in rat focal cerebral ischemia models. However, the mechanisms by which microglia are neuroprotective remain unclear. This study evaluated whether post-ischemic treatment with another integrin αvβ3 inhibitor, the cyclic arginine-glycine-aspartic acid (RGD) peptide-cGRGDdvc (LXW7), alleviates cerebral ischemic injury. The anti-inflammatory effect of LXW7 in activated microglia within rat focal cerebral ischemia models was examined. A total of 108 Sprague-Dawley rats (250–280 g) were subjected to middle cerebral artery occlusion (MCAO). After 2 h, the rats were given an intravenous injection of LXW7 (100 μg/kg) or phosphate-buffered saline (PBS). Neurological scores, infarct volumes, brain water content (BWC) and histology alterations were determined. The expressions of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)], and Iba1-positive activated microglia, within peri-ischemic brain tissue, were assessed with ELISA, western blot and immunofluorescence staining. Infarct volumes and BWC were significantly lower in LXW7-treated rats compared to those in the MCAO + PBS (control) group. The LXW7 treatment lowered the expression of pro-inflammatory cytokines. There was a reduction of Iba1-positive activated microglia, and the TNF-α and IL-1β expressions were attenuated. However, there was no difference in the Zea Longa scores between the ischemia and LXW7 groups. The results suggest that LXW7 protected against focal cerebral ischemia and attenuated inflammation in activated microglia. LXW7 may be neuroprotective during acute MCAO-induced brain damage and microglia-related neurodegenerative diseases.

Recombinant adenovirus expressing hIFN-λ1 inhibits gastric adenocarcinoma cell line SGC-7901 proliferation.

The aim of the present study is to investigate the effect of a recombinant plasmid adenovirus (pAd) expressing human interferon-λ1 (hIFN-λ1) on the proliferation of the gastric adenocarcinoma cell line SGC-7901. For this purpose, human gastric adenocarcinoma SGC-7901 cells were infected with recombinant pAd-hIFN-λ1, pAd-LacZ and phosphate-buffered saline (PBS), respectively, and the subsequent effects on the proliferation of the infected cells were compared. Cell proliferation was evaluated by MTT assay, while mRNA and protein expression of hIFN-λ1 were detected by reverse transcription-polymerase chain reaction analysis and immunofluorescence assay, respectively. In addition, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay and flow cytometry were conducted to analyze the rate of cell apoptosis. The results indicated that the proliferation of gastric adenocarcinoma SGC-7901 cells was significantly inhibited by pAd-hIFN-λ1. Furthermore, the apoptosis rate and the mRNA and protein expression levels of hIFN-λ1 were higher in pAd-hIFN-λ1-transfected cells, compared with the pAd-LacZ and PBS control groups. In conclusion, recombinant pAd-hIFN-λ1 induced the expression of hIFN-λ1 in gastric adenocarcinoma SGC-7901 cells, and significantly inhibited cell proliferation by promoting apoptosis in these cancer cells.

Spatiotemporal PET Imaging of Dynamic Metabolic Changes After Therapeutic Approaches of Induced Pluripotent Stem Cells, Neuronal Stem Cells, and a Chinese Patent Medicine in Stroke.

This study aimed to use spatiotemporal PET imaging to investigate the dynamic metabolic changes after a combined therapeutic approach of induced pluripotent stem cells (iPSCs), neuronal stem cells (NSCs), and Chinese patent medicine in a rat model of cerebral ischemia-reperfusion injury. Cerebral ischemia was established by the middle cerebral artery occlusion approach. Thirty-six male rats were randomly assigned to 1 of the 6 groups: control phosphate-buffered saline (PBS), Chinese patent medicine (Qing-kai-ling [QKL]), induced pluripotent stem cells (iPSCs), combination of iPSCs and QKL, neuronal stem cells (NSCs), and combination of NSCs and QKL. Serial (18)F-FDG small-animal PET imaging and neurofunctional tests were performed weekly. Autoradiographic imaging and immunohistochemical and immunofluorescent analyses were performed at 4 wk after stem cell transplantation. Compared with the PBS control group, significantly higher (18)F-FDG accumulations in the ipsilateral cerebral infarction were observed in 5 treatment groups from weeks 1-4. Interestingly, the most intensive (18)F-FDG accumulation was found in the NSCs + QKL group at week 1 but in the iPSCs + QKL group at week 4. The neurofunctional scores in the 5 treatment groups were significantly higher than that of the PBS group from week 3 to 4. In addition, there was a significant correlation between the PET imaging findings and neurofunctional recovery (P < 0.05) or glucose transporter-1 expression (P < 0.01). Immunohistochemical and immunofluorescence studies found that transplanted iPSCs survived and migrated to the ischemic region and expressed protein markers for cells of interest. Spatiotemporal PET imaging with (18)F-FDG demonstrated dynamic metabolic and functional recovery after iPSCs or NSCs combined with QKL in a rat model of cerebral ischemia-reperfusion injury. iPSCs or NSCs combined with Chinese medicine QKL seemed to be a better therapeutic approach than these stem cells used individually.

Prediction of T cell epitopes of Brucella abortus and evaluation of their protective role in mice.

Brucellae are Gram-negative intracellular bacteria that cause an important zoonotic disease called brucellosis. The animal vaccines are available but have disadvantage of causing abortions in a proportion of pregnant animals. The animal vaccines are also pathogenic to humans. Recent trend in vaccine design has shifted to epitope-based vaccines that are safe and specific. In this study, efforts were made to identify MHC-I- and MHC-II-restricted T cell epitopes of Brucella abortus and evaluate their vaccine potential in mice. The peptides were designed using online available immunoinformatics tools, and five MHC-I- and one MHC-II-restricted T cell peptides were selected on the basis of their ability to produce interferon gamma (IFN-γ) in in vivo studies. The selected peptides were co-administered with poly DL-lactide-co-glycolide (PLG) microparticles and evaluated for immunogenicity and protection in BALB/c mice. Mice immunized with peptides either entrapped in PLG microparticles (EPLG-Pep) or adsorbed on PLG particles (APLG-Pep) showed significantly higher splenocyte proliferation and IFN-γ generation to all selected peptides than the mice immunized with corresponding irrelevant peptides formulated PLG microparticles or phosphate-buffered saline (PBS). A significant protection compared to PBS control was also observed in EPLG-Pep and APLG-Pep groups. A plasmid DNA vaccine construct (pVaxPep) for peptides encoding DNA sequences was generated and injected to mice by in vivo electroporation. Significant protection was observed (1.66 protection units) when compared with PBS and empty vector control group animals. Overall, the MHC-I and MHC-II peptides identified in this study are immunogenic and protective in mouse model and support the feasibility of peptide-based vaccine for brucellosis.

Onset Time and Durability of Huntingtin Suppression in Rhesus Putamen After Direct Infusion of Antihuntingtin siRNA.

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27–39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification.

The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

Utility and Safety of Commercially Available Injection Laryngoplasty Materials in a Rabbit Model

To demonstrate foreign body and chronic inflammatory reaction of commercially available injection materials using the rabbit vocal fold paralysis model. Animal study. The left recurrent laryngeal nerve was identified and divided at the tracheoesophageal groove. Amounts (100μL) of phosphate-buffered saline (PBS), polyacrylamide hydrogel (Aquamid; Ferrosan A/S, Søborg, Denmark), calcium hydroxyapatite (Radiesse; BioForm Medical Inc., San Mateo, CA), or hyaluronic acid derivative (Rofilan; Rofil Medical International, Breda, Netherlands) were injected into the left vocalis muscle. Six months later, the larynx was harvested. Hematoxylin/eosin and Masson trichrome staining were performed to compare inflammatory and foreign body reactions, granuloma development, and relative vocal fold areas among groups. Compared with the PBS (control) group, the Aquamid, Radiesse, and Rofilan groups exhibited only mild chronic inflammatory reactions that did not significantly differ among groups, or from controls (P>0.05). However, the Aquamid and Radiesse groups exhibited moderate foreign body reactions that were significantly greater than those of controls (P<0.05). No foreign body granuloma formed in any group. All test groups exhibited significant increases in vocal fold areas at 6months (P<0.05). Although commercially available injection materials induced more foreign body reactions than a control injection of PBS, no foreign body granuloma developed and the augmented vocal fold area was maintained until 6months after injection.

Intranasal administration of a combination of choline chloride, vitamin C, and selenium attenuates the allergic effect in a mouse model of airway disease

Respiratory allergic disease is an inflammatory condition accompanied by oxidative stress. Supplementation of an anti-inflammatory agent with antioxidants may have a therapeutic effect. In this study, the effects of choline chloride in combination with antioxidants were evaluated via the intranasal route in a mouse model of allergic airway disease. Balb/c mice were sensitized on days 0, 7, and 14 and challenged on days 25–30 with cockroach extract (CE) and with a booster challenge on day 38. They were treated with choline chloride (ChCl; 1mg/kg), vitamin C (Vit C; 308.33mg/kg), and selenium (Se; 1mg/kg) alone or in combination via the intranasal route on days 31, 33, 35, 37, and 39. The mice were sacrificed on day 40 to collect blood, bronchoalveolar lavage fluid, lungs, and spleen. Mice immunized with CE showed a significant increase in airway hyperresponsiveness (AHR), lung inflammation, Th2 cytokines, and the oxidative stress markers intracellular reactive oxygen species and 8-isoprostanes compared to the phosphate-buffered saline control group. A significant decrease was observed in these parameters with all the treatments (p<0.01). The highest decrease was noticed in the ChCl+Vit C+Se-treated group, with AHR decreased to the normal level. This group also showed the highest decrease in airway inflammation (p<0.001), IL-4 and IL-5 (p<0.001), IgE and IgG1 (p<0.001), NF-κB (p<0.001), and 8-isoprostane levels (p<0.001). Glutathione peroxidase activity, which was decreased significantly in CE-immunized mice, was restored to normal levels in this group (p<0.001). IL-10 level was decreased in CE-immunized mice and was restored to normal by combination treatment. The combination treatment induced FOXP3+ cells in splenocyte culture, responsible for the upregulation of IL-10. In conclusion, the combination of choline chloride, vitamin C, and selenium via the intranasal route reduces AHR, inflammation, and oxidative stress, probably by causing IL-10 production by FOXP3+ cells, and possesses therapeutic potential against allergic airway disease.

Toll-Like Receptor 7 Agonist Imiquimod in Combination with Influenza Vaccine Expedites and Augments Humoral Immune Responses against Influenza A(H1N1)pdm09 Virus Infection in BALB/c Mice

Toll-like receptors (TLRs) of the innate immune system are known targets for enhancing vaccine efficacy. We investigated whether imiquimod, a synthetic TLR7 agonist, can expedite the immune response against influenza virus infection when combined with influenza vaccine. BALB/c mice were immunized intraperitoneally with monovalent A(H1N1)pdm09 vaccine combined with imiquimod (VCI) prior to intranasal inoculation with a lethal dose of mouse-adapted A(H1N1)pdm09 virus. For mice immunized 3 days before infection, the survival rates were significantly higher in the VCI group (60%, mean survival time[MST], 11 days) than in the vaccine-alone (30%; MST, 8.8 days), imiquimod-alone (5%; MST, 8.4 days), and phosphate-buffered saline (PBS) (0%; MST, 6.2 days) groups (P < 0.01). In the VCI group, 45 and 35% of the mice survived even when they were infected 2 days or 1 day after immunization. Virus-specific serum IgM, IgG, and neutralizing antibodies appeared earlier with higher geometric mean titers in the VCI group than in the control groups. The pulmonary viral load was significantly lower at all time points postinfection in the VCI, vaccine-alone, and imiquimod-alone groups than in the PBS control group (P < 0.05). The protection induced by VCI was specific for A(H1N1)pdm09 virus but not for A(H5N1) virus. Since imiquimod combined with RNase-treated vaccine is as protective as imiquimod combined with untreated vaccine, mechanisms other than TLR7 may operate in expediting and augmenting immune protection. Moreover, increased gamma interferon mRNA expression and IgG isotype switching, which are markers of the Th1 response induced by imiquimod, were not apparent in our mouse model. The mechanisms of imiquimod-induced immune protection deserve further study.

Immunological response to bolus versus multiple injections of hylan G‐F 20 (Synvisc®) in a murine biocompatibility model

Local tissue reactivity to intra-articular injections of hyaluronic acid hylan G-F 20 (Synvisc) has been described. We used a murine biocompatibility model to study the inflammatory response to Synvisc after a single bolus injection versus the traditional three shot series of injections. Air pouches were established subcutaneously in BALB/c mice, which were injected with phosphate-buffered saline (PBS), 5 mg ultra-high molecular weight polyethylene particles (to simulate synthetic joint wear debris, positive control), 0.5 mL Synvisc (one injection/week for three weeks, harvested 14 days after last injection), or 1.5 mL Synvisc bolus (harvested either 14 or 28 days after last injection). Inflammatory gene expression and inflammation of air pouch tissue, and serum antibody titers to Synvisc were determined. Inflammation was observed with all Synvisc treatments relative to PBS (p < 0.01). However, the three injection series of Synvisc resulted in significantly (p < 0.05) greater tumor necrosis factor-alpha gene expression compared to both PBS and bolus single shot Synvisc harvested after 14 or 28 days. While all Synvisc treatments resulted in serum antibodies to Synvisc (p < 0.02 compared to PBS control group), mice that received three injections of Synvisc had higher levels than mice receiving a single injection (p < 0.01). These results demonstrate that a single bolus injection of Synvisc led to less inflammation and a lower antibody response when compared to the three-shot series of injections, supporting the current change in treatment from multiple injections to a single injection of Synvisc .

ENOS Overexpressing Bone Marrow Cells are Safe and Effective in a Porcine Model of Myocardial Regeneration Following Acute Myocardial Infarction

Cell therapy has been shown to be effective in improving LV function postmyocardial infarction (MI). We hypothesized that eNOS-transfected bone marrow cells (BMCs) are safe in a swine model of myocardial infarction (MI). We also hypothesized that endothelial nitric oxide synthase (eNOS) transfection would enhance cell function, as assessed by myocardial functional recovery post-MI. Fifteen female Yorkshire pigs underwent bone marrow aspiration and creation of MI. Bone marrow cells were cultured for 7days, and each pig received either autologous BMCs transiently transfected with eNOS plasmid (eNOS-BMC, n=5), nontransfected BMCs (nt-BMC, n=4), or phosphate-buffered saline (PBS) control (n=6). Cardiac MRI was performed at baseline (1week post-MI) and 6weeks post-MI. There was no difference in safety outcomes between groups. Absolute left ventricular ejection fraction (LVEF) at 6weeks showed a trend toward improvement in both cell therapy groups compared with baseline but worsened in the PBS control group. The absolute improvement in LVEF was significantly greater in both cell therapy groups compared with PBS control. Infarct mass was significantly lower in the eNOS-BMC group between baseline and 6weeks, but the absolute change in infarct mass was not different between groups. Finally, there was a trend toward reduced LV mass in the eNOS-BMC group. Bone marrow cell delivery, with and without eNOS overexpression, is safe and leads to improvement in LVEF when administered in the coronary circulation 7days following acute MI in swine. Transfection of healthy BMCs with eNOS resulted in some improvement in left ventricular remodeling. Further study is warranted in a preclinical model that approximates the impact of cardiovascular risk factors on BMC function.

Naïve rat umbilical cord matrix stem cells significantly attenuate mammary tumor growth through modulation of endogenous immune responses

Background aimsUn-engineered human and rat umbilical cord matrix stem cells (UCMSCs) attenuate growth of several types of tumors in mice and rats. However, the mechanism by which UCMSCs attenuate tumor growth has not been studied rigorously. MethodsThe possible mechanisms of tumor growth attenuation by rat UCMSCs were studied using orthotopic Mat B III rat mammary tumor grafts in female F344 rats. Tumor-infiltrating leukocytes were identified and quantified by immunohistochemistry analysis. Potential cytokines involved in lymphocyte infiltration in the tumors were determined by microarray and Western blot analysis. The Boyden chamber migration assay was performed for the functional analysis of identified cytokines. ResultsRat UCMSCs markedly attenuated tumor growth; this attenuation was accompanied by considerable lymphocyte infiltration. Immunohistochemistry analysis revealed that most infiltrating lymphocytes in the rat UCMSC-treated tumors were CD3+ T cells. In addition, treatment with rat UCMSCs significantly increased infiltration of CD8+ and CD4+ T cells and natural killer (NK) cells throughout tumor tissue. CD68+ monocytes/macrophages and Foxp3+ regulatory T cells were scarcely observed, only in the tumors of the phosphate-buffered saline control group. Microarray analysis of rat UCMSCs demonstrated that monocyte chemotactic protein-1 is involved in rat UCMSC-induced lymphocyte infiltration in the tumor tissues. ConclusionsThese results suggest that naïve rat UCMSCs attenuated mammary tumor growth at least in part by enhancing host anti-tumor immune responses. Naïve UCMSCs can be used as powerful therapeutic cells for breast cancer treatment, and monocyte chemotactic protein-1 may be a key molecule to enhance the effect of UCMSCs at the tumor site.

Inhibitory Effect of Adenoviral Vector-Mediated Delivery of p21WAF1/CIP1 on Retinal Vascular Endothelial Cell Proliferation and Tube Formation in Cultured Rhesus Monkey Cells (RF/6A)

Objective: To investigate the inhibitory effect(s) of adenovirus (Ad) vector-mediated delivery of p21WAF1/CIP1 (Ad-p21) on proliferation and tube formation in Rhesus monkey choroid-retina vascular endothelial cells (RF/6A).Methods: In vitro-cultured RF/6A cells were divided into three groups: phosphate-buffered saline (PBS), Ad-p21-transfected, and negative control. Plasmid vectors were transfected via Ad-p21. The mRNA and protein expressions of p21 and cyclin-dependent kinase (CDK)2 in RF/6A cells were measured by reverse transcription-PCR (RT-PCR) and western blot analyses. Cell-cycle distributions were analyzed by flow cytometry. Matrigel was used as a matrix for endothelial cell tube formation.Results: Expressions of p21 mRNA and protein were greater, and expressions of CDK2 mRNA and protein lower, in the Ad-p21-transfected group than in either the PBS or negative control groups. Cell-cycle distribution analysis indicated that the proportion of G0/G1 cells was significantly higher in the Ad-p21 transfected group than in either the PBS or negative control groups (p = 0.000). There were significantly fewer endothelial cell tubes in the Ad-p21-transfected group than in either the PBS or negative control groups (p = 0.004).Conclusions: Ad-p21 inhibits RF/6A cell proliferation and tube formation. The underlying mechanism to account for this may be that overexpression of p21 arrests the cell-cycle transition from the G1- to the S-phase via inhibition of CDK2 activity.

Efficacy of immunization with the recombinant collagen adhesin A region against Staphylococcus aureus -induced mouse mastitis

Staphylococcus aureus (S. aureus) is a major cause of bovine mastitis. In this present study, we assessed the efficacy of the recombinant A region of collagen adhesin (rCna-A) as a mastitis vaccine in a mouse model. Three groups of mice were immunized with either rCna-A, inactivated S. aureus, or phosphate-buffered saline (PBS) as a control. IgG and IgG subtype titers of mice in the rCna-A vaccine group were significantly higher than those in the killed vaccine group (P<0.01). Immunized lactating mice were challenged with S. aureus via the intramammary route. Significantly, fewer bacteria were recovered from mice in the rCna-A group than from those in the killed vaccine group (P<0.001). Histopathology indicated that the mammary structure showed greater integrity and a milder inflammatory response in the rCna-A group as compared with both the inactivated vaccine and PBS control groups. These results suggested that the rCna-A protein may be an attractive target for a vaccine against S. aureus-induced mastitis. Key words: Staphylococcus aureus, collagen adhesion, inactivated vaccine, mastitis.

An Adjuvanted Herpes Simplex Virus 2 Subunit Vaccine Elicits a T Cell Response in Mice and Is an Effective Therapeutic Vaccine in Guinea Pigs

Immunotherapeutic herpes simplex virus 2 (HSV-2) vaccine efficacy depends upon the promotion of antigen-specific immune responses that inhibit reactivation or reactivated virus, thus controlling both recurrent lesions and viral shedding. In the present study, a candidate subunit vaccine, GEN-003/MM-2, was evaluated for its ability to induce a broad-spectrum immune response in mice and therapeutic efficacy in HSV-2-infected guinea pigs. GEN-003 is comprised of HSV-2 glycoprotein D2 (gD2ΔTMR340-363) and a truncated form of infected cell polypeptide 4 (ICP4383-766), formulated with Matrix M-2 (MM-2) adjuvant (GEN-003/MM-2). In addition to eliciting humoral immune responses, CD4(+) and CD8(+) T cells characterized by the secretion of multiple cytokines and cytolytic antigen-specific T cell responses that were able to be recalled at least 44 days after the last immunization were induced in immunized mice. Furthermore, vaccination with either GEN-003 or GEN-003/MM-2 led to significant reductions in both the prevalence and severity of lesions in HSV-2-infected guinea pigs compared to those of phosphate-buffered saline (PBS) control-vaccinated animals. While vaccination with MM-2 adjuvant alone decreased recurrent disease symptoms compared to the PBS control group, the difference was not statistically significant. Importantly, the frequency of recurrent viral shedding was considerably reduced in GEN-003/MM-2-vaccinated animals but not in GEN-003- or MM-2-vaccinated animals. These findings suggest a possible role for immunotherapeutic GEN-003/MM-2 vaccination as a viable alternative to chronic antiviral drugs in the treatment and control of genital herpes disease.

Hyaluronan Protects Bovine Articular Chondrocytes Against Cell Death Induced by Bupivacaine at Supraphysiologic Temperatures

Background: Bupivacaine and supraphysiologic temperature can independently reduce cell viability of articular chondrocytes. In combination, these 2 deleterious factors could further impair cell viability. Hypothesis: Hyaluronan may protect chondrocytes from death induced by bupivacaine at supraphysiologic temperatures. Study Design: Controlled laboratory study. Methods: Bovine articular chondrocytes were treated with hyaluronan at physiologic (37°C) and supraphysiologic temperatures (45°C and 50°C) for 1 hour and then exposed to bupivacaine for 1 hour at room temperature. Cell viability was assessed at 3 time points: immediately after treatment, 6 hours later, and 24 hours later using flow cytometry and fluorescence microscopy. The effects of hyaluronan on the levels of sulfated glycosaminoglycan in the chondrocytes were determined using Alcian blue staining. Results: (1) Bupivacaine alone did not induce noticeable chondrocyte death at 37°C; (2) bupivacaine and temperature synergistically increased chondrocyte death, that is, when the chondrocytes were conditioned to 45°C and 50°C, 0.25% and 0.5% bupivacaine increased the cell death rate by 131% to 383% in comparison with the phosphate-buffered saline control group; and (3) addition of hyaluronan reduced chondrocyte death rates to approximately 14% and 25% at 45°C and 50°C, respectively. Hyaluronan’s protective effects were still observed at 6 and 24 hours after bupivacaine treatment at 45°C. However, at 50°C, hyaluronan delayed but did not prevent the cell death caused by bupivacaine. One-hour treatment with hyaluronan significantly increased sulfated glycosaminoglycan levels in the chondrocytes. Conclusion: Bupivacaine and supraphysiologic temperature synergistically increase chondrocyte death, and hyaluronan may protect articular chondrocytes from death caused by bupivacaine. Clinical Relevance: This study provides a rationale to perform preclinical and clinical studies to evaluate whether intra-articular injection of a mixture of bupivacaine and hyaluronan after arthroscopic surgery may protect against bupivacaine’s chondrotoxicity.

Detrimental effects of rat mesenchymal stromal cell pre-treatment in a model of acute kidney rejection

Mesenchymal stromal cells (MSC) have shown immunomodulatory and tissue repair potential including partial tolerance induction by pre-treatment of donor-specific cells in a rat heart transplantation model. Very recently, we could show that autologous MSC attenuated ischemia reperfusion injury in a highly mismatched donor–recipient rat kidney transplant model. Therefore, we investigated donor-specific MSC pre-treatment in this rat kidney transplantation model to study whether graft function could be improved, or if tolerance could be induced. Donor- and recipient-type MSC or phosphate buffered saline (PBS) as a control was injected i.v. 4 days before kidney transplantation. Mycophenolate mofetil immunosuppression (20mg/kg body weight) was applied for 7 days. Kidney grafts and spleens were harvested between days 8 and 10 and analyzed by quantitative RT-PCR and immunohistology. In addition, creatinine levels in the blood were measured and serum was screened for the presence of donor-specific antibodies. Surprisingly, application of both donor- and recipient-specific MSC resulted in enhanced humoral immune responses verified by intragraft B cell infiltration and complement factor C4d deposits. Moreover, signs of inflammation and rejection were generally enhanced in both MSC-treated groups relative to PBS control group. Additionally, pre-treatment with donor-specific MSC significantly enhanced the level of donor-specific antibody formation when compared with PBS- or recipient MSC-treated groups. Pre-treatment with both MSC types resulted in a higher degree of kidney cortex tissue damage and elevated creatinine levels at the time point of rejection. Thus, MSC pre-sensitization in this model impairs the allograft outcome. Our data from this pre-clinical kidney transplantation model indicate that pre-operative MSC administration may not be optimal in kidney transplantation and caution must be exerted before moving forward with clinical studies in order to avoid adverse effects.

2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

Expression of microRNA-146a in lipopolysaccharide challenged alveolar macrophages in vitro

To investigate the expression of microRNA-146a (miRNA-146a) in NR8383 alveolar macrophages treated with lipopolysaccharides (LPS). NR8383 alveolar macrophages were divided into two groups: LPS treated group and phosphate buffer saline (PBS) control group, and they cultured for 6 hours. The production of tumor necrosis factor-α (TNF-α) in the supernatant of cells was determined with enzyme-linked immunosorbent assay (ELISA), and the expression of miRNA-146a of cells was detected by real-time polymerase chain reaction (PCR). Compared with PBS control group, the TNF-α content (ng/L) in LPS treated group was significantly increased (650.26±40.53 vs. 6.23±1.76, P<0.01), and miRNA-146a in LPS treated group increased by about (5.33±0.81) folds (P<0.01). The expression of miRNA-146a was increased in LPS treated NR8383 cells, and miRNA-146a may be involved in the modulation inflammatory response of the NR8383 alveolar macrophage.

The influence of exogenous cross-linking and compressive creep loading on intradiscal pressure

This study involves a biomechanical evaluation of a prospective injectable treatment for degenerative discs. The high osmolarity of the non-degenerated nucleus pulposus attracts water contributing to the hydrostatic behavior of the tissue. This intradiscal pressure is known to drop as fluid is exuded from the matrix due to compressive loading. The objective of this study was to compare the changes in intradiscal pressure in control and genipin cross-linked intervertebral discs. Thirty bovine lumbar motion segments were randomly divided into a phosphate-buffered saline control group and a 0.33% genipin group and soaked at room temperature for 2 days. A needle pressure sensor was held in the center of the disc while short-term and static creep compressive loads were applied. The control group demonstrated a 25% higher average intradiscal pressure compared to genipin-treated discs under 750 N compressive load (p=0.029). Depressurization during static compressive creep was 56% higher in the control than in the genipin group (p=0.014). These results suggest cross-linking induced changes in the poroelastic properties of the involved tissues affected the mechanics of compressive load support in the disc with lower levels of nucleus pressure, a corresponding decrease in the elastic expansion of the annulus, and an increased axial compressive loading of the inner and outer annulus tissues. It is possible that concurrent changes in hydraulic permeability and proteoglycan retention known to be associated with genipin cross-linking were also contributors to poroelastic changes. Reduction of peak pressures and moderation of pressure fluctuations could be beneficial relative to discogenic pain.