Number Of Green Fluorescent Protein Research Articles

Intraocular Axon Regeneration in a Model of Penetrating Eye Injury.

Purpose: Penetrating eye injuries commonly cause permanent loss of vision in patients. Unlike mammals, zebrafish can regenerate both damaged tissue and severed axons in the central nervous system. Here, we present a tractable adult zebrafish model to study intraocular axon regeneration after penetrating eye injury. Methods: To create consistent penetrating intraocular injuries, pins of standardized diameters were inserted into the eye through the cornea and penetrating the retina but not the underlying sclera. Transgenic gap43:GFP reporter fish were used to preferentially label retinal ganglion cells (RGCs) that respond to injury with regenerating axons. Retinas were fixed and flat mounted at various times postinjury to examine injury size, number of green fluorescent protein (GFP)-positive cells and axons, axonal varicosities, and rate of regeneration to the optic nerve head. Intraocular injection of colchicine was used to inhibit axon outgrow as a proof of principle that this method can be used to screen effects of pharmacological agents on intraocular axon regeneration. Results: Penetrating injury to the zebrafish retina results in robust axon regeneration by RGCs around and beyond the site of injury. The gap43:GFP transgene allows visualization of individual or small bundles of axons with varicosities and growth cones easily observable. Regeneration proceeded with most, if not all, axons reaching the optic nerve head by 3-day postinjury. A single intraocular injection of colchicine a day after injury was sufficient to delay axon regeneration at 2-days postinjury. Surprisingly, we identified a stereotypically located population of circumferential projecting neurons within the retina that upregulate gap43:GFP expression after injury. Conclusions: Penetrating injury to the adult gap43:GFP transgenic zebrafish eye is a model of successful intraocular axon regeneration. The pharmacological and genetic tools available for this organism should make it a powerful tool for dissecting the cellular, molecular, and genetic mechanisms of axon regeneration in the intraocular environment.

Neural stem cells transplantation combined with ethyl stearate improve PD rats motor behavior by promoting NSCs migration and differentiation.

In recent years, the ability of neural stem cells (NSCs) transplantation to treat Parkinson's disease (PD) has attracted attention. However, it is still a challenge to promote the migration of NSCs to the lesion site and their directional differentiation into dopaminergic neurons in PD. C-C motif chemokine ligand 5 (CCL5) and C-C motif chemokine receptor 5 (CCR5) are expressed in the brain and are important regulators of cell migration. It has been reported that ethyl stearate (PubChem CID: 8122) has a protective effect in 6-OHDA-induced PD rats. Parkinson's disease rats were injected with 6-hydroxydopamine (6-OHDA) into the right substantia nigra, and striatum followed by 8μL of an NSC cell suspension containing 100 μM ethyl stearate and 8 × 105 cells in the right striatum. The effect of transplantation NSCs combined with ethyl stearate was assessed by evaluating apomorphine (APO)-induced turning behavior and performance in the pole test. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting (WB), and immunofluorescence staining were also performed. NSCs transplantation combined with ethyl stearate ameliorated the behavioral deficits of PD rats. PD rats that received transplantation NSCs combined with ethyl stearate exhibited increased expression of tyrosine hydroxylase (TH) and an increased number of green fluorescent protein (GFP)-positive cells. Furthermore, GFP-positive cells migrated into the substantia nigra and differentiated into dopaminergic neurons. The expression of CCL5 and CCR5 was significantly increased after transplantation NSCs combined with ethyl stearate. These findings suggest that NSCs transplantation combined with ethyl stearate can improve the motor behavioral performance of PD rats by promoting NSCs migration from the striatum to the substantia nigra via CCL5/CCR5 and promoting the differentiation of NSCs into dopaminergic neurons.

In Situ-Formed Fibrin Hydrogel Scaffold Loaded With Human Umbilical Cord Mesenchymal Stem Cells Promotes Skin Wound Healing

Healing of full-thickness skin wounds remains a major challenge. Recently, human umbilical cord mesenchymal stem cells (hUC-MSCs) were shown to possess an extraordinary potential to promote skin repair in clinical settings. However, their low survival rate after transplantation limits their therapeutic efficiency in treating full-thickness skin wounds. Hydrogels are considered an ideal cell transplantation vector owing to their three-dimensional mesh structure, good biosafety, and biodegradation. The objective of this study was to investigate the skin wound healing effect of a fibrin hydrogel scaffold loaded with hUC-MSCs. We found that the fibrin hydrogel had a three-dimensional mesh structure and low cytotoxicity and could prolong the time of cell survival in the peri-wound area. The number of green fluorescent protein (GFP)-labeled hUC-MSCs was higher in the full-thickness skin wound of mice treated with hydrogel–hUC-MSCs than those of mice treated with cell monotherapy. In addition, the combination therapy between the hydrogel and hUC-MSCs speed up wound closure, its wound healing rate was significantly higher than those of phosphate-buffered saline (PBS) therapy, hydrogel monotherapy, and hUC-MSCs monotherapy. Furthermore, the results showed that the combination therapy between hydrogel and hUC-MSCs increased keratin 10 and keratin 14 immunofluorescence staining, and upregulated the relative gene expressions of epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA), promoting epithelial regeneration and angiogenesis. In conclusion, the fibrin hydrogel scaffold provides a relatively stable sterile environment for cell adhesion, proliferation, and migration, and prolongs cell survival at the wound site. The hydrogel–hUC-MSCs combination therapy promotes wound closure, re-epithelialization, and neovascularization. It exhibits a remarkable therapeutic effect, being more effective than the monotherapy with hUC-MSCs or hydrogel.

Controlling soft rot of postharvest chilli pepper (Capsicum annuum L.) by an antagonist Bacillus amyloliquefaciens S917: Efficacy and action mode

Pectobacterium carotovorum, the causal agent of soft rot, is responsible for significant economic damage in storage and transportation of postharvest chilli pepper. In this study, an antagonist S917, identified as Bacillus amyloliquefaciens based on the 16S rDNA sequence, was screened from 1400 strains for antagonistic activity towards Pectobacterium carotovorum subsp. brasiliense XC1 (Pcb XC1). The in vitro antibacterial result showed that cells and peptide extract of B. amyloliquefaciens S917 had good inhibitory activity against Pcb XC1. Based on draft genome sequences of B. amyloliquefaciens S917, five bacteriocins (Thusin_alpha, Thusin_beta2, Amylocyclicin, ComX1, LCI-like) were identified by BAGEL4 database and four antibacterial metabolites (Bacillaene, Macrolactin H, Bacilysin, Bacillibactin) were identified by antiSMASH database. The in vivo disease control result of chilli pepper showed that cells of B. amyloliquefaciens S917 performed better in reducing the rotting incidence and lesion diameter. Result of gas chromatography-ion mobility spectrometry (GC-IMS) showed that B. amyloliquefaciens S917 cells (BSC) treatment protected the original volatile aroma of chilli pepper. However, the control had richer volatile compounds related to soft rot, including methylbutanoate, dimethyl sulfide, methylbutanal, 3-methyl-2-butenal, pentan-1-ol, 3-methylbutan-1-ol, hexanol. Further, action mode of BSC was investigated. The number of green fluorescent protein labelled Pcb XC1 in chilli pepper was reduced by more than 99% after BSC treatment. Result of scanning electron microscopy also showed that BSC treatment prevented the damage of pepper epidermal structure caused by Pcb XC1. Moreover, the result of RNA-seq showed that BSC treatment triggered defense responses of chilli pepper. Pathways of plant pathogen interaction, phenylpropanoid biosynthesis and metabolism, plant hormone synthesis and signal transduction, MAPK signaling, WRKY transcription factor, and cell wall synthesis were activated. This study confirmed that B. amyloliquefaciens S917 can be used as an antagonist to control soft rot of postharvest chilli pepper.

Brain injury triggers cell-type-specific and time-dependent endoplasmic reticulum stress responses.

The unfolded protein response (UPR) is a signal transduction network that responds to endoplasmic reticulum (ER) stress by coordinating protein homeostasis to maintain cell viability. The UPR can also trigger cell death when adaptive responses fail to improve protein homeostasis. Despite accumulating evidence suggesting that the UPR plays a role in neurodegenerative diseases and brain insults, our understanding of how ER stress is induced under neuropathological conditions is limited. Here, we investigated the cell- and time-specific patterns of the ER stress response after brain injury using ER stress-activated indicator (ERAI) mice, which enable monitoring of the UPR in vivo via increased fluorescence of a spliced XBP-1 protein fused with the green fluorescent protein (GFP) variant Venus. Following cortical stab injury of ERAI mice, the GFP signal and number of GFP+ cells increased in the ipsilateral cortex throughout the observation period (6h to 7 days post-injury), confirming the induction of the UPR. GFP signals were observed in injured neurons early (from 6h) after brain injury. However, non-neuronal cells, mainly endothelial cells followed by astrocytes, accounted for the majority of GFP+ cells after brain injury. Similar results were obtained in a mouse model of focal cerebral ischemia. These findings suggest that activation of the UPR in both neuronal and non-neuronal cells, especially endothelial cells and astrocytes, may play an important role in and could be a potential therapeutic target for acute brain injuries.

Fibrotic remodeling of left ventricle in the murine remnant kidney model of chronic kidney disease is independent of arterial hypertension

Abstract Background Cardiac fibrosis and arterial hypertension are common in patients with chronic kidney disease (CKD). We studied the mechanisms of cardiac fibrogenesis and the role of blood pressure in mice with CKD. Methods and results 10-week-old male C57/BL6N (BL6) and SV129 wildtype (WT) mice were underwent 5/6 nephrectomy (remnant kidney model, RKM) or sham operation for 10 weeks. RKM significantly elevated plasma creatinine and urea. RKM elicited both interstitial and replacement renal and left ventricular (LV) (BL6: SHAM 5.6±0.4%, RKM 7.3±0.7%, p=0.04; SV129: SHAM 6.0±0.5%, RKM 14±2%, p=0.001) fibrosis as assessed by picrosirius red staining. In parallel, the number of cardiac fibroblasts per mm2 (BL6: SHAM 36±4, RKM 85±13, p=0.001; SV129: SHAM 82±11, RKM 200±34, p=0.006) was increased in RKM mice. With regard to possible mechanisms, cardiac oxidative stress as shown by co-immunostaining for intracellular fibronectin and 8-hydroxyguanosine (BL6: SHAM 44±14%, RKM 60±24%, p=0.03; SV129: SHAM 51±6%, RKM 70±7%, p=0.04) and the percentage of CXCR4+ fibroblasts in the myocardium (BL6: SHAM 47±5%, RKM 62±4%, p=0.04; SV129: SHAM 63±5%, RKM 81±3%, p=0.005) were increased. Furthermore, the number of circulating CD45+ / collagen I+ fibrocytes (FACS) in the peripheral blood was increased by RKM in BL6 (SHAM 100±23%, RKM 443±252%, p=0.04) and diminished in SV129 (SHAM 100±19%, RKM 43±11%, p=0.01), while an opposite regulation was seen in the bone marrow. To further confirm the role of bone-marrow derived fibroblasts in renal and cardiac remodeling 10-week-old WT BL6 mice were subjected to transplantation of bone marrow from 10-week-old WT BL6 mice expressing green fluorescent protein (GFP)+ ubiquitously. 28 days later, RKM or SHAM-operation was performed. RKM significantly increased the number of GFP+ fibroblasts in kidney and LV-myocardium. CKD significantly decreased myocardial capillarization assessed by immunostaining for podocalyxin in both mouse lines. In parallel, myocardial protein expression of fibrosis regulators fibronectin, collagen I, CTGF and Hif1a were up-regulated and expression of the active form of eNOS (phospho-S1177) was reduced. As a possible confounder, tail-cuff blood pressure was moderately enhanced (Ø 30mmHg) 9 weeks after nephrectomy. In a control experiment using the vasodilator hydralazine (250 mg/L/day), peripheral blood pressure was equalized in all 4 experimental groups, but the extent of LV fibrosis and expression of the above-mentioned fibrosis markers remained unchanged. Conclusions Chronic kidney disease in the RKM model elicits left ventricle fibrosis by increasing myocardial protein expression of fibrosis regulators, reduction of myocardial capillarization and mobilization / recruitment of circulating fibroblasts, independently of blood pressure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Deutsche Forschungsgemeinschaft

Single Molecule Fluorescence Imaging Reveals the Stoichiometry of BKγ1 Subunit in Living HEK293 Cell Expression System

Large conductance Ca2+-activated K+ (BKCa) channels are ubiquitously expressed in plasma membrane of both excitable and non-excitable cells and possess significant physiological functions. A tetrameric complex of α subunit (BKα) forms a functional pore of BKCa channel. The properties of BKCa channel, such as voltage-dependence, Ca2+ sensitivity and pharmacological responses, are extensively modulated by co-expressing accessory β subunits (BKβ), which can associate with BKα in one to one manner. Although the functional significance of newly identified γ subunits (BKγ) has been revealed, the stoichiometry between BKα and BKγ1 remains unclear. In the present study, we utilized a single molecule fluorescence imaging with a total internal reflection fluorescence (TIRF) microscope to directly count the number of green fluorescent protein (GFP)-tagged BKγ1 (BKγ1-GFP) within a single BKCa channel complex in HEK293 cell expression system. BKγ1-GFP significantly enhanced the BK channel activity even when the intracellular Ca2+ concentration was kept lower, i.e., 10 nM, than the physiological resting level. BKγ1-GFP stably formed molecular complexes with BKα-mCherry in the plasma membrane. Counting of GFP bleaching steps revealed that a BKCa channel can contain up to four BKγ1 per channel at the maximum. These results suggest that BKγ1 forms a BKCa channel complex with BKα in a 1 : 1 stoichiometry in a human cell line.

α‐Hederin induce gastric cancer cell death by blocking autophagic flux and triggering ER stress

BackgroundGastric cancer (GC) is a prevalent refractory disease worldwide, especially in developing countries. α‐Hederin (α‐Hed) is a major active component of triterpenoid saponin isolated from Fructus Akebia, we found it showed significant anti‐proliferative and pro‐apoptotic activity against GC cells in a dose‐dependent manner.PurposeThis study aims to uncover the potential mechanism of α‐Hedin relation to anti‐GC activity.Study designCytotoxicity of a‐Hed to BGC‐823 and MGC‐803 cells was monitored by CCK8 and Annexin V‐FITC/PI apoptosis assay. Autophagic flux was examined by western blot analysis and the number of green fluorescent protein (GFP)‐LC3B puncta. ER Stress was also probed by western blot analysis. Cellular Reactive Oxygen Species (ROS) Detection Assay Kit was used to detect ROS level.ResultsGC cells growth was significantly inhibited and cell death was induced by α‐Hed in BGC‐823 and MGC‐803 cells. We subsequently discovered that α‐Hed promoted autophagosome formation by upregulatingthe expression of LC3‐II, Beclin‐1 and increasing GFP‐LC3B puncta formation. However, p62 was also upregulated, which was indicative of autophagic flux blockage. Comparing to α‐Hed treatment alone, the combinatorial treatment of α‐Hed and Bafilomycin A1 (Baf A1) did not obviously alter the expression of LC3‐II, suggest that α‐Hed blocked autophagic flux in GC cells. Nevertheless it was later discovered that expression of ATF6, IRE1α, and PERK were elevated after a‐Hed treatment in GC cells, which represents activation of three signaling arms of the Unfolded Protein Response. Besides, α‐Hed obviously increased cellular ROS levels in GC cells, and the ER stress‐regulated apoptosis related proteins such as CHOP, caspase‐12, and JNK also increased.ConclusionOur results suggest that α‐Hed induce GC cell death by influencing crosstalk between autophagy and ER Stress pathways.Support or Funding InformationNational Natural Science Foundation of China General Program (81973523)graphical abstractFigure 1

Trabecular meshwork mesenchymal stem cell transplantation improve motor symptoms of parkinsonian rat model

Stem cell transplantation is a new therapeutic strategy in the treatment of neurodegenerative disorders such as Parkinson's disease (PD). Therefore, in this study, the therapeutic effects of Trabecular Meshwork Mesenchymal Stem Cells (TM-MSCs) transplantation, as a new source of mesenchymal stem cells, were evaluated in the animal model of PD.After the development and confirmation of hemi-parkinsonian rats by administration of 6-hydroxy dopamine (6-OHDA) and apomorphine-induced rotation test, green fluorescent protein (GFP) labeled TM-MSCs (normal and induced cells) were transplanted in the striatum of rats. Next, the rotation test, rotarod test, open field, passive avoidance memory tests and immunohistochemistry for tyrosine hydroxylase (TH) were done.The results showed that the number of turns significantly decreased and the improvement of motor performance was achieved after cell transplantation. However, there was no significant difference in passive avoidance memory of animals documented by shuttle box test. The number of GFP- labeled cells expressing TH significantly is increased compared to the vehicle group.Collectively, it seems that TM-MSCs and induced TM-MSCs cell transplantation have positive effects on some aspects of the animal model of PD. Other studies may reveal the potentially positive aspects of these cells in the laboratory and clinical studies.

Effects of Hippo signaling pathway on lung injury repair by mesenchymal stem cells in acute respiratory distress syndrome

To investigate the effects of Hippo signaling pathway on lung injury repair of mesenchymal stem cells (MSC) in acute respiratory distress syndrome (ARDS) and its mechanism. Mouse bone marrow-derived MSC (mMSCs) cell lines with low expression of large tumor suppressor 2 (LATS2) were constructed by lentiviral vector transfection. Male C57BL/6 mice aging 6-8 weeks old were divided into four groups according to random number table (n = 36). The ARDS animal model (ARDS group) was reproduced by intratracheally injection of 2 g/L lipopolysaccharide (LPS) 50 μL, the normal saline (NS) control group was injected with an equal volume of NS. After 4 hours of model reproduction, 5×104 mMSCs transfected with blank lentivirus vector (MSC-shcontrol group) or shLATS2 lentivirus vector (MSC-shLATS2 group) were transplanted intratracheally, while NS control group and ARDS group were injected with equal volume of phosphate buffered saline (PBS). Mice were sacrificed at 3, 7, and 14 days after modeling, and lung tissue and bronchoalveolar lavage fluid (BALF) were harvested. Near-infrared fluorescence imaging, immunofluorescence staining and Western Blot were used to track mMSCs in lung tissue. Retension and proportion of mMSC differentiation into type II alveolar epithelial cells (AEC II) were evaluated. Lung tissue wet weight/body weight ratio (LWW/BW) and total protein (TP) and albumin (ALB) in BALF were determined to reflect pulmonary edema. The expression of Occludin protein in lung epithelium was tested by Western Blot to reflect permeability of epithelium. The levels of interleukins (IL-1β, IL-6, IL-10) in BALF were assessed by enzyme-linked immunosorbent assay (ELISA) to reflect lung inflammation. Hematoxylin-eosin (HE) staining and modified Masson staining were carried out, and the scores were assessed to reflect lung injury and evaluate pulmonary fibrosis. The signal intensity of isolated lung fluorescence images at 3 days in the MSC-shLATS2 group was significantly higher than that in the MSC-shcontrol group (fluorescence intensity: 0.039±0.005 vs. 0.017±0.002, P < 0.05), the number of green fluorescent protein (GFP)-positive cells in lung tissue was also significantly higher than that in the MSC-shcontrol group (cells/HP: 29.65±6.98 vs. 17.50±4.58, P < 0.05), but they all decreased with time; and the proportion of mMSCs differentiated into AEC II was significantly increased [(64.12±15.29)% vs. (19.64±3.71)%, P < 0.05]. Compared with the NS control group, the levels of surface active protein C (SPC) and Occludin protein in the ARDS group were significantly decreased, LWW/BW ratio and TP, ALB and inflammatory factors levels in BALF were significantly increased; extensive alveolar and interstitial edema, hemorrhage and diffuse inflammatory cell infiltration were found in lung tissue, and the lung injury score was significantly increased; collagen fibers were deposited in alveolar septum and alveolar cavity, and pulmonary fibrosis score was also increased significantly. Compared with the ARDS group, the expression levels of SPC and Occludin at 14 days in the MSC-shcontrol group and the MSC-shLATS2 group were significantly increased (SPC/β-actin: 0.51±0.12, 0.68±0.10 vs. 0.27±0.08, Occludin/β-actin: 0.49±0.19, 0.79±0.11 vs. 0.25±0.08, all P < 0.05), TP, ALB, IL-1β, IL-6 levels in BALF at 3 days were significantly decreased [TP (g/L): 8.08±1.72, 5.12±0.87 vs. 12.55±2.09; ALB (g/L): 0.71±0.21, 0.44±0.18 vs. 1.18±0.29, IL-1β (ng/L): 99.26±14.32, 60.11±8.58 vs. 161.86±25.17, IL-6 (ng/L): 145.54±13.29, 101.74±11.55 vs. 258.79±27.88, all P < 0.05], and IL-10 was significantly increased (ng/L: 190.83±22.61, 316.65±37.88, both P < 0.05). Furthermore, all the above parameters in the MSC-shLATS2 group were significantly improved as compared with those in the MSC-shcontrol group (all P < 0.05). LWW/BW ratio in the MSC-shLATS2 group was significantly lower than that in the ARDS group and the MSC-shcontrol group (mg/g: 9.85±1.51 vs. 16.78±1.92, 14.88±1.74, both P < 0.05). Inhibiting Hippo signaling pathway by low expression of LATS2 could promote the retention of mMSCs in lung tissue and differentiation into AEC II cells of ARDS mice, improve pulmonary edema and alveolar epithelial permeability, regulate pulmonary inflammatory response, and alleviate pathological damage and fibrosis of lung tissue.

The immune influence of a parabiosis model on tumour-bearing mice.

The aim of this study was to analyse the immune influence of a parabiosis model on tumour-bearing mice. Parabiosis was established between C57BL/6 wild-type mice expressing green fluorescent protein (GFP+) and C57BL/6 wild-type mice without green fluorescent protein (GFP) to ensure blood cross-circulation between animals, and then the expression of CD4+ T cells, CD8+ T cells and interleukins 2, 4 and 10, and interferon-gamma (INF-γ) in spleen cells of parabiosis model mice were examined with flow cytometry. At day 8 and day 14 after conjoined surgery, we were aiming to sample tumour tissue in the parabiosis mice and observe changes of CD3, CD4, CD8, CD31, IFN-γ and vascular endothelial growth factor (VEGF) through immunohistochemical analysis. The interaction of blood was established on the third day with modelling rate of 85.7% after blood interaction. The healthy cells of GFP+ C57 mice entered the blood circulation of tumour-bearing mice via a connecting capillary network, playing a role in stimulating CD4+ and CD8+ cells in the tumour-bearing mice so that CD4+ cells increased more in tumour-bearing mice than in the positive control group (p <0.05). The number of GFP+ cells that were detected in a tumour-bearing mouse was small, but GFP+ cells can stimulate the mouse itself to generate more CD4+/interleukin (IL)-4, CD4+/IL-10 (p <0.05).The numbers of CD4+/IL-2, CD4+/IL-4 and CD4+/IL-10 among the GFP+ mice were higher than those in the negative control group(p <0.05).The levels of IFN-γ in both mice in the parabiosis model were decreased (p <0.05). The rate of CD4+/CD8+ in parabiosis GFP+ mice was higher than in the negative control group (p <0.05). In immunohistochemical tests, the rates of CD3, CD4, CD8 and IFN-γ positive cells was higher than in the positive control group, with their optical densities of 0.32 ± 0.63, 0.33 ± 0.00, 0.31 ± 0.91 and 0.28 ± 0.14 respectively (p <0.05). The expression of CD31 (0.19 ± 0.50) and VEGF (0.19 ± 0.21) were lower when compared with the positive control group, with no significant difference. CD31 and VEFG cell expression was low, at 0.19 ± 0.50 and 0.19 ± 0.21, respectively, compared with the positive control group (p >0.05). Values for CD31 and VEGF cells in the positive control group were higher, at 0.32 ± 0.35 and 0.29 ± 0.35, respectively, but when compared with the parabiosis tumour-bearing group, there was no significant difference. The expression of CD3, CD4, CD8 and IFN-γ cells at day 8 was low: 0.22, 0.17, 0.15 and 0.16, respectively. When compared with the parabiosis tumour-bearing group, there was no significant difference. The established allogeneic parabiosis mice model can be well adapted to the conjoined state of mice and be applied in wide medical experiments. The parabiosis model has played an important role in studying immune regulation, which provides a basis for the future tumour immunotherapy. Parabiosis models can stimulate tumour-bearing mice to generate CD3, CD4, CD8 and IFN-γ, and play a notable role in immune regulation and tumour destruction. The positive expression rates of CD31 and VEFG cells in the parabiosis tumour-bearing group were lower; however, when compared with the positive control group, there was no significant difference.

Trapped in the epineurium: early entry into the endoneurium is restricted to neuritogenic T cells in experimental autoimmune neuritis

BackgroundAutoimmune polyneuropathies are acquired inflammatory disorders of the peripheral nervous system (PNS) characterized by inflammation, demyelination, and axonal degeneration. Although the pathogenesis has not been fully elucidated, T cells recognizing self-antigens are believed to initiate inflammation in a subgroup of patients. However, the route and time of T cell entry into the PNS have not yet been described in detail. In this study, we analyzed both kinetics as well as localization of retrovirally transfected green fluorescent protein (GFP)-expressing neuritogenic T lymphocytes in experimental autoimmune neuritis (EAN).MethodsT lymphocytes obtained from rats following EAN induction by immunization with peripheral nerve protein peptide P255–78 were retrovirally engineered to express GFP. Non-specific T cells were negatively selected by in vitro restimulation, whereas GFP-expressing neuritogenic T cells (reactive to P255–78) were adoptively transferred into healthy rats (AT-EAN). Antigen-specific T cell tracking and localization was performed by flow cytometry and immunohistochemistry during the course of disease.ResultsAfter induction of autoimmune neuritis, P2-reactive T cells were detectable in the liver, spleen, lymph nodes, lung, peripheral blood, and the sciatic nerves with distinct kinetics. A significant number of GFP+ T cells appeared early in the lung with a peak at day four. In the peripheral nerves within the first days, GFP-negative T cells rapidly accumulated and exceeded the number of GFP-expressing cells, but did not enter the endoneurium. Very early after adoptive transfer, T cells are found in proximity to peripheral nerves and in the epineurium. However, only GFP-expressing neuritogenic T cells are able to enter the endoneurium from day five after transfer.ConclusionsOur findings suggest that neuritogenic T cells invade the PNS early in the course of disease. However, neuritogenic T cells cross the blood-nerve barrier with a certain delay without preference to dorsal roots. Further understanding of the pathophysiological role of autoagressive T cells may help to improve therapeutic strategies in immune-mediated neuropathies.

Fusion of GFP and phase contrast images with complex shearlet transform and Haar wavelet-based energy rule.

Image fusion techniques can integrate the information from different imaging modalities to get a composite image which is more suitable for human visual perception and further image processing tasks. Fusing green fluorescent protein (GFP) and phase contrast images is very important for subcellular localization, functional analysis of protein and genome expression. The fusion method of GFP and phase contrast images based on complex shearlet transform (CST) is proposed in this paper. Firstly the GFP image is converted to IHS model and its intensity component is obtained. Secondly the CST is performed on the intensity component and the phase contrast image to acquire the low-frequency subbands and the high-frequency subbands. Then the high-frequency subbands are merged by the absolute-maximum rule while the low-frequency subbands are merged by the proposed Haar wavelet-based energy (HWE) rule. Finally the fused image is obtained by performing the inverse CST on the merged subbands and conducting IHS-to-RGB conversion. The proposed fusion method is tested on a number of GFP and phase contrast images and compared with several popular image fusion methods. The experimental results demonstrate that the proposed fusion method can provide better fusion results in terms of subjective quality and objective evaluation.

Production of GFP and Luciferase-Expressing Reporter Macrophages for In Vivo Bioluminescence Imaging.

Macrophages have emerged as crucial regulators of tissue homeostasis, inflammation, and tissue regeneration. In vivo bioluminescence imaging could offer a powerful tool to study many poorly understood aspects of macrophage biology. Thus, we recently developed a straightforward method for the production of large numbers of green fluorescent protein (GFP) and firefly luciferase (fLUC)-expressing reporter macrophages for various in vivo bioluminescence imaging applications. Lentivirus vector containing the GFP/fLUC reporter gene is produced and mouse bone marrow macrophages are isolated following established protocols. Macrophages are then exposed to the lentivirus in the presence of 10μM cyclosporine for 24h. After a 24-h recovery period, the transduction is repeated. Three days after the second infection the cells are ready to be used in vivo. Following this cyclosporine-mediated double infection strategy up to 60% of the macrophages express GFP in flow cytometry. The macrophages maintain their ability to polarize to M1 and M2 phenotypes and, when injected to the systemic circulation of a mouse model, reporter cells are both easily detectable with BLI and migrate to a local site of inflammation. These GFP/fLUC-expressing reporter macrophages could prove to be useful tools to study the role of macrophages in health and disease.

Trichostatin A resistance is facilitated by HIF-1α acetylation in HeLa human cervical cancer cells under normoxic conditions.

Trichostatin A (TSA) is an anticancer drug that inhibits histone deacetylases (HDACs). Hypoxia-inducible factor 1 (HIF-1) participates in tumor angiogenesis by upregulating target genes, such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether TSA treatment increases HIF-1α stabilization via acetylation under normoxic conditions, which would lead to VEGF upregulation and resistance to anticancer drugs. TSA enhanced total HIF-1α and VEGF-HRE reporter activity under normoxic conditions. When cells were transfected with GFP-HIF-1α, treatment with TSA increased the number of green fluorescence protein (GFP)-positive cells. TSA also enhanced the nuclear translocation of HIF-1α protein, as assessed by immunoblotting and as evidenced by increased nuclear localization of GFP-HIF-1α. An increase in the interaction between HIF-1α and the VEGF promoter, which was assessed by a chromatin immunoprecipitation (ChIP) assay, led to activation of the VEGF promoter. TSA acetylated HIF-1α at lysine (K) 674, which led to an increase in TSA-induced VEGF-HRE reporter activity. In addition, TSA-mediated cell death was reduced by the overexpression of HIF-1α but it was rescued by transfection with a HIF-1α mutant (K674R). These data demonstrate that HIF-1α may be stabilized and translocated into the nucleus for the activation of VEGF promoter by TSA-mediated acetylation at K674 under normoxic conditions. These findings suggest that HIF-1α acetylation may lead to resistance to anticancer therapeutics, such as HDAC inhibitors, including TSA.

Recruitment of bone marrow-derived cells to the periodontal ligament via the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 axis.

The periodontal ligament (PDL) is a non-mineralized connective tissue that exists between the alveolar bone and root surface cementum and plays important roles in tooth function. The PDL harbors a remarkable reserve of multipotent stem cells, which maintain various types of cells. However, the sources of these stem cells, other than their developmental origin, are not well understood. To elucidate the recruitment of bone marrow (BM)-derived stem cells in the PDL, green fluorescent protein (GFP)-expressing BM-derived cells were transplanted into the femoral BM of immunodeficient rats, and the distribution and expression of stem cell markers in the PDL were analyzed in vivo. To evaluate the functional significance of BM-derived cells to the PDL, tooth replantation was performed and the expression of stromal cell-derived factor (SDF)-1, a critical chemotactic signal for mesenchymal stem cell recruitment, was analyzed. To confirm the SDF-1-dependency of BM-derived cell migration to the PDL, PDL-conditioned medium (CM) was prepared, and BM-derived cell migration was analyzed using a transwell culture system. Four weeks after cell transplantation, GFP-positive cells were detected in the PDL, and some of them were also positive for stem cell markers (i.e., CD29, SSEA4, and αSMA). Seven days after tooth replantation, the number of GFP- and SDF-1-positive cells significantly increased in PDL. Concurrently, the concentration of SDF-1 and the number of colony-forming units of fibroblasts in peripheral blood were increased. BM-derived cell migration increased in PDL-CM and was inhibited by an inhibitor of C-X-C chemokine receptor type 4 (CXCR4), an SDF-1 receptor. These results indicate that stem cells and their progeny in PDL are not only derived from their developmental origin but are also supplied from the BM via the blood as the need arises. Moreover, this BM-derived cell recruitment appears to be regulated, at least partially, by the SDF-1/CXCR4 axis.

Effect of Naoxintong capsule on endothelial progenitor cell mobilization and homing following bone marrow transplantation in a mouse hind limb ischemia model

Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells. Signal molecules produced by ischemia and hypoxia can promote mobilization of bone marrow EPCs to peripheral circulation and formation of novel blood vessels in tissues that are damaged during heart attack. Naoxintong capsule (NXT) has the functions of promoting blood circulation, removing blood stasis, promoting the circulation of qi and relieving pain. The various components in NXT have protective effects on blood vessels and can effectively improve the symptoms of ischemia. However, its effect on EPCs is not clear. To study the intervention effect of NXT on mobilization and homing of peripheral blood EPCs, green fluorescent protein (GFP) transgenic mice were used for bone marrow transplantation (BMT) and then unilateral hind limb ischemia model (UHLI) were constructed. For BMT, wild-type ICR mice were irradiated by CS137 and then injected with 4×106 bone marrow cells isolated from GFP mice. The bone marrow reconstitution of recipients was assessed by quantification of GFP bone marrow-derived cells (BMDC) from transplanted mice 4 weeks after BMT. The UHLI model was duplicated by ligating femoral artery and divided into three groups: the model group, the NXT group (model+NXT) and the positive control group (model+simvastatin). Flow cytometry was used to detect the proportion of GFP positive cells and the peripheral blood EPCs levels at 1, 3, 7, 14 days before and after surgery. Ischemic tissue of gastrocnemius muscle was excised at 3 and 7 days after operation for immunofluorescence staining to detect the number of GFP+ cells. The bone marrow chimerism was achieved at day 28 after BMT. There was no significant difference in the percentage of GFP positive cells between BMT mice and GFP transgenic mice. NXT and simvastatin could significantly increase the number of peripheral blood EPCs 1,3 days after surgery. Three and seven days after operation, the number of homing EPCs was significantly higher in NXT group and positive control group than that in model group (P<0.001). In conclusion, NXT can obviously promote the mobilization and homing of EPCs.

Change in number and size of circulating tumor cells with high telomerase activity during treatment of patients with gastric cancer.

Detection of circulating tumor cells (CTCs) in peripheral blood is useful for estimating the prognosis of patients with cancer. We previously reported the detection of CTCs by OBP-401, a telomerase-specific, replication-selective, oncolytic adenoviral agent carrying the green fluorescent protein (GFP) gene. We demonstrated that the number of large (L)-GFP+ cells (≥7.735 µm in diameter) in peripheral blood samples correlated significantly with the prognosis of treatment-naïve gastric cancer patients, whereas the number of small (S)-GFP+ cells (<7.735 µm in diameter) did not. In the present study, we studied the change in the number of GFP+ cells during treatment, and analyzed the association between the number of GFP+ cells in blood samples and the outcome of patients. Peripheral blood samples were obtained from 37 gastric patients prior and subsequent to surgery (three samples per time point). Upon infection of blood cells with OBP-401, GFP+ cells of different sizes were counted and measured. The association between the number of GFP+ cells and surgical outcome was determined by statistical analysis. The median follow-up period after surgery was 39 months. Although the difference was not significant, patients with ≥6 L-GFP+ cells in preoperative blood samples had a lower relapse-free survival rate than patients with 0-5 L-GFP+ cells. There was no significant correlation between the number of L-GFP+ cells in postoperative blood samples and the prognosis of patients receiving adjuvant therapy. Although the difference was not significant, the number of S-GFP+ cells in samples from patients who had received postoperative chemotherapy was higher than in those who had not. The number of L-GFP+ cells was not significantly correlated with the relapse-free survival rate in gastric cancer patients who underwent surgery. The number of S-GFP+ cells was relatively high in samples from patients who had received postoperative chemotherapy.

A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells.

We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.

Autophagy is deficient in nasal polyps: implications for the pathogenesis of the disease

Nasal polyposis is characterized by persistent inflammation but the pathogenesis is complex and still debatable. Autophagy has been associated with many human health problems including chronic inflammatory airway diseases. Whether autophagy plays a role in nasal polyps and could be a therapeutic target is completely unknown. We studied light chain 3 (LC3) protein expression, a common indication of autophagy, in fresh tissue specimens of 5 nasal polyps and 6 normal nasal mucosa by Western blot analysis. The results were also confirmed by immunohistochemistry (IHC) using additional 25 paraffin-embedded nasal tissue sections. Finally the autophagic activity was validated in nasal polyp-derived fibroblasts by evaluating the number of green fluorescent protein (GFP)-labeled LC3 puncta. The expression of LC3 was dramatically decreased in all 5 nasal polyp tissues. In contrast, protein kinase B-mechanistic target of rapamycin (Akt-mTOR) signaling, an established negative regulator of autophagy, was significantly activated in these tissues. Immunohistochemical results further demonstrated a negative correlation between autophagy and nasal polyps (p < 0.05). GFP-LC3 puncta formation, an alternative indicator of autophagy, was also diminished in nasal polyp-derived fibroblasts (p < 0.01). Autophagy is deficient presumably due to suppression by high Akt-mTOR activity in nasal polyps, which may provide a molecular basis for future mechanistic study of the disease.