Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc

Introduction

The neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been identified in the human intervertebral disc (IVD) and have been implicated in the mechanisms associated with nerve ingrowth and nociception in degeneration of the IVD. The aim of the current study was to investigate an association between neurotrophin expression in the IVD and the severity of disc degeneration, including the effect of disc-related proinflammatory cytokines on neurotrophin and neuropeptide expression in cells derived from the human IVD.

Methods

Immunohistochemical analysis was performed to examine the expression of NGF, BDNF and their high-affinity receptors Trk-A and Trk-B in human IVD samples, divided into three categories: non-degenerate, moderate degeneration and severe degeneration. In order to study the effect of disc-related cytokines on neurotrophin/neuropeptide gene expression, nucleus pulposus cells derived from non-degenerate and degenerate IVD samples were seeded in alginate and were stimulated with either IL-1β or TNFα for 48 hours. RNA was extracted, cDNA was synthesised and quantitative real-time PCR was performed to examine the expression of NGF, BDNF and substance P.

Results

Immunohistochemistry showed expression of NGF and BDNF in the native chondrocyte-like cells in all regions of the IVD and in all grades of degeneration. Interestingly only BDNF significantly increased with the severity of degeneration (P < 0.05). Similar expression was observed for Trk-A and Trk-B, although no association with disease severity was demonstrated. In cultured human nucleus pulposus cells, stimulation with IL-1β led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFα was associated with an upregulation of substance P expression only.

Conclusion

Our findings show that both the annulus fibrosus and nucleus pulposus cells of the IVD express the neurotrophins NGF and BDNF, factors that may influence and enhance innervation and pain in the degenerate IVD. Expression of Trk-A and Trk-B by cells of the nondegenerate and degenerate IVD suggests an autocrine role for neurotrophins in regulation of disc cell biology. Furthermore, modulation of neurotrophin expression by IL-1β and modulation of substance P expression by TNFα, coupled with their increased expression in the degenerate IVD, highlights novel roles for these cytokines in regulating nerve ingrowth in the degenerate IVD and associated back pain.     

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

Introduction

The degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.

Methods

Quantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.

Results

Initial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.

Conclusions

The release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.     

CCL20 Secretion from the Nucleus Pulposus Improves the Recruitment of CCR6-Expressing Th17 Cells to Degenerated IVD Tissues

Background

Studies elucidated that Th17 cells are important contributors to the pathogenesis of many immune-mediated diseases, and IL-17A is present in pathologic intervertebral disc (IVD) tissues. However, the mechanisms, how these cells traffic into the degenerate discs are not clear.

Materials and Methods

The samples collected from 53 patients had been divided into 3 groups: Group P (annulus fibrosus was intact), Group E (annulus fibrosus was reptured) and normal control. Immunohistochemistry was used to detect the expression of CCL20, CCR6, IL-17A, TNF–α and CD4 in IVD tissues. Moreover, nucleus pulposus (NP) cells had been cultured in the presence and absence of Th17 associated cytokines. The supernatants were detected for CCL20 concentrations by ELISA, and the NP cells for the expression of CCL20 mRNA. Additionally, peripheral blood (PB) samples had undergone detection for the expression of CCR6 mRNA and the proportion of IL-17-producing cells, including the surface expression of CCR6.

Results

Immunohistochemistry revealed that CCL20 and TNF-α were expressed in degenerated NP cells. Double-labeled immunofluorescence elaborated, IL-17-producing cells (CD4⁺IL-17A⁺ and CD4⁺CCR6⁺) appeared in the Group E samples, but no traces or expression in Group P and normal control. IL-17A and TNF-α, alone or combined, could enhance CCL20 secretion in a dose-dependent manner, which was obtained through RT-PCR results. There was a notable difference of CCR6 mRNA expression between patients and normal controls. In comparison to controls, flow cytometry data indicated that the proportion of IL-17-producing cells and the CCR6 expression in PB were significantly increased.

Conclusion

Our results provide a potential explanation for involvement of the CCL20-CCR6 system in the trafficking of IL-17-producing cells to degenerated IVD tissues. Additionally, our results explain the contribution of Th17 associated cytokines to the development of degenerated discs via the up-regulation of CCL20 secretion from NP cells, which forms a positive chemotactic feedback loop.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

Hyaluronan injection in murine osteoarthritis prevents TGFbeta 1-induced synovial neovascularization and fibrosis and maintains articular cartilage integrity by a CD44-dependent mechanism

Introduction

The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling.

Methods

Rat nucleus pulposus cells were cultured under normoxic (21% O₂) or hypoxic (2% O₂) conditions, and the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated ??-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells.

Results

Nucleus pulposus cells exhibited increased ??-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of ??-catenin gene and protein compared with untreated control cells.

Conclusions

These data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.     

The chemokine,CXCL16, and its receptor,CXCR6, are constitutively expressed in human annulus fibrosus and expression of CXCL16 is up-regulated by exposure to IL-1ß in vitro

Chemokines are an important group of soluble molecules with specialized functions in inflammation. The roles of many specialized chemokines and their receptors remain poorly understood in the human intervertebral disc. We investigated CXCL16 and its receptor, CXCR6, to determine their immunolocalization in disc tissue and their presence following exposure of cultured human annulus fibrosus cells to proinflammatory cytokines. CXCL16 is a marker for inflammation; it also can induce hypoxia-inducible factor 1α (HIF-1α), which is a phenotypic marker of heathy nucleus pulposus tissue. We found CXCL16 and CXCR6 immunostaining in many cells of the annulus portion of the disc. Molecular studies showed that annulus fibrosus cells exposed to IL-1ß, but not TNF-α, exhibited significant up-regulation of CXCL16 expression vs. control cells. There was no significant difference in the percentage of annulus cells that exhibited immunolocalization of CXCL16 in grade I/II, grade III or grade IV/V specimens. The presence of CXCL16 and its receptor, CXCR6, in the annulus in vivo suggests the need for future research concerning the role of this chemokine in proinflammatory functions, HIF-1α expression and disc vascularization.     

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Introduction

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.

Methods

Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).

Results

The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.

Conclusion

We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.     

Disruption of the thrombospondin-2 gene alters the lamellar morphology but does not permit vascularization of the adult mouse lumbar disc

Introduction

The biological basis for the avascular state of the intervertebral disc is not well understood. Previous work has suggested that the presence of thrombospondin-1 (TSP-1), a matricellular protein, in the outer annulus reflects a role for this protein in conferring an avascular status to the disc. In the present study we have examined thrombospondin-2 (TSP-2), a matricellular protein with recognized anti-angiogenic activity in vivo and in vitro.

Methods

We examined both the location and expression of TSP-2 in the human disc, and its location in the disc and bordering soft tissues of 5-month-old normal wild-type (WT) mice and of mice with a targeted disruption of the TSP-2 gene. Immunohistochemistry and quantitative histology were utilized in this study.

Results

TSP-2 was found to be present in some, but not all, annulus cells of the human annulus and the mouse annulus. Although there was no difference in the number of disc cells in the annulus of TSP-2-null mice compared with that of WT animals, polarized light microscopy revealed a more irregular lamellar collagen structure in null mouse discs compared with WT mouse discs. Additionally, vascular beds at the margins of discs of TSP-2-null mice were substantially more irregular than those of WT animals. Counts of platelet endothelial cell adhesion molecule-1-positive blood vessels in the tissue margin bordering the ventral annulus showed a significantly larger vascular bed in the tissue bordering the disc of TSP-2-null mice compared with that of WT mice (P = 0.0002). There was, however, no vascular ingrowth into discs of the TSP-2-null mice.

Conclusion

These data confirm a role for TSP-2 in the morphology of the disc and suggest the presence of other inhibitors of angiogenesis in the disc. We have shown that although an increase in vasculature was present in the TSP-2-null tissue in the margin of the disc, vascular ingrowth into the body of the disc did not occur. Our results point to the need for future research to understand the transition from the well-vascularized status of the fetal and young discs to the avascular state of the adult human disc or the small mammalian disc.     

Expression of cartilage-derived morphogenetic protein in human intervertebral discs and its effect on matrix synthesis in degenerate human nucleus pulposus cells

Introduction  

Loss of intervertebral disc (IVD) matrix and ultimately disc height as a result of 'degeneration' has been implicated as a major cause of low back pain (LBP). The use of anabolic growth factors as therapies to regenerate IVD matrix, hence restoring disc height and thus reversing degenerative disc disease, has been suggested. Cartilage-derived morphogenetic protein (CDMP) is a growth factor which stimulates proteoglycan production in chondrocyte-like cells and thus could be a useful growth factor for LBP therapies. However, little is known about the expression of CDMP or its receptor in human IVD, nor its effects on human disc cells.     

Acute ST-segment elevation myocardial infarction after amoxycillin-induced anaphylactic shock in a young adult with normal coronary arteries: a case report

Background

Sirolimus-eluting stents (CYPHER stents) demonstrated remarkable efficacy in reducing restenosis rates in patients with coronary artery disease. There is a concern of sub-acute and late stent thrombosis. Tissue factor (TF) is critical in thrombosis. This study investigated the effect of sirolimus on TF expression and activity in cultured human vascular smooth muscle cells (SMCs).

Methods

SMCs were cultured from human saphenous veins and aortas. Quiescent cells were stimulated with sirolimus (0.1 – 20 ng/ml) over 24 hours. Cellular TF expression and activity released into culture medium were measured. The effect of sirolimus on activation of mammalian target of rapamycin (mTOR) was measured by phosphorylation of the substrate p70s6k at T389, and activation of RhoA was measured by pull-down assay.

Results

Sirolimus increased TF protein level in cultured human SMCs in a concentration and time-dependent manner (about 2-fold, p < 0.01) reaching maximal effect at 5 ng/ml. The stimulation of TF expression by sirolimus was associated with inhibition of basal activity of mTOR. No effects of sirolimus on RhoA or p38mapk activation that are positive regulators of TF in vascular wall cells were observed. The stimulation of TF expression by sirolimus (20 ng/ml) was prevented by the HMG-CoA reductase inhibitor fluvastatin (1 μmol/L). However, no increase in TF activity released from SMC into culture medium was observed after sirolimus treatment.

Conclusion

Although sirolimus stimulates TF protein expression in human SMC associated with inhibition of mTOR, it does not enhance TF activity released from the cells, suggesting a relatively safe profile of CYPHER stents. The inhibition of TF expression by fluvastatin favors clinical use of statins in patients undergoing coronary stenting.     

Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

Background

The founding member of the EMAP-like protein family is the Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs. The EMAP-like protein family has five members in mammals (EML1 through EML5) and only one in both Drosophila (ELP-1) and C. elegans (ELP-1). Biochemical studies of sea urchin EMAP and vertebrate EMLs implicate these proteins in the regulation of microtubule stability. So far, however, the physiological function of this protein family remains unknown.

Results

We examined the expression pattern of C. elegans ELP-1 by means of transgenic gene expression in living embryos and adults, and by immunolocalization with an ELP-1-specific antibody in fixed tissues. In embryos, ELP-1 is expressed in the hypodermis. In larvae and adults, ELP-1 is expressed in the body wall, spermatheca and vulval muscles, intestine, and hypodermal seam cells. In muscle, ELP-1 is associated with adhesion complexes near the cell surface and is bound to a criss-crossing network of microtubules in the cytoplasm. ELP-1 is also expressed in a subset of mechanoreceptor neurons, including the ray neurons in the male tail, microtubule-rich touch receptor neurons, and the six ciliated IL1 neurons. This restricted localization in the nervous system implies that ELP-1 plays a role in mechanotransmission. Consistent with this idea, decreasing ELP-1 expression decreases sensitivity to gentle touch applied to the body wall.

Conclusion

These data imply that ELP-1 may play an important role during the transmission of forces and signals between the body surface and both muscle cells and touch receptor neurons.     

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Introduction  

Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.     

A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

Introduction

Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.

Methods

Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.

Results

TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.

Conclusions

Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

Effect of glutathione reductase knockdown in response to UVB-induced oxidative stress in human lung adenocarcinoma

Background

Glutathione reductase (GR) plays a critical role in the maintenance of physiological redox status in cells. However, the comprehensive investigations of GR-modulated oxidative stress have not been reported.

Methods

In the present study, we cultured a human lung adenocarcinoma line CL1-0 and its GR-knockdown derivative CL1-0ΔGR to evaluate their differential responses to UVB-irradiation.

Results

We identified 18 proteins that showed significant changes under UVB-irradiation in CL1-0ΔGR cells rather than in CL1-0 cells. Several proteins involving protein folding, metabolism, protein biosynthesis and redox regulation showed significant changes in expression.

Conclusions

In summary, the current study used a comprehensive lung adenocarcinoma-based proteomic approach for the identification of GR-modulated protein expression in response to UVB-irradiation. To our knowledge, this is the first global proteomic analysis to investigate the role of GR under UVB-irradiation in mammalian cell model.     

Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D

Background

Patients with asthma demonstrate circadian variations in the airway inflammation and lung function. Pinealectomy reduces the total inflammatory cell number in the asthmatic rat lung. We hypothesize that melatonin, a circadian rhythm regulator, may modulate the circadian inflammatory variations in asthma by stimulating the chemotaxins expression in the lung epithelial cell.

Methods

Lung epithelial cells (A549) were stimulated with melatonin in the presence or absence of TNF-α(100 ng/ml). RANTES (Regulated on Activation Normal T-cells Expressed and Secreted) and eotaxin expression were measured using ELISA and real-time RT-PCR, eosinophil chemotactic activity (ECA) released by A549 was measured by eosinophil chemotaxis assay.

Results

TNF-α increased the expression of RANTES (307.84 ± 33.56 versus 207.64 ± 31.27 pg/ml of control, p = 0.025) and eotaxin (108.97 ± 10.87 versus 54.00 ± 5.29 pg/ml of control, p = 0.041). Melatonin(10^-10 to 10^-6M) alone didn't change the expression of RNATES (204.97 ± 32.56 pg/ml) and eotaxin (55.28 ± 6.71 pg/ml). However, In the presence of TNF-α (100 ng/ml), melatonin promoted RANTES (410.88 ± 52.03, 483.60 ± 55.37, 559.92 ± 75.70, 688.42 ± 95.32, 766.39 ± 101.53 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7,10^-6M melatonin, respectively) and eotaxin (151.95 ± 13.88, 238.79 ± 16.81, 361.62 ± 36.91, 393.66 ± 44.89, 494.34 ± 100.95 pg/ml, treated with 10^-10, 10^-9, 10^-8, 10^-7, 10^-6M melatonin, respectively) expression in a dose dependent manner in A549 cells (compared with TNF-α alone, P < 0.05). The increased release of RANTES and eotaxin in A549 cells by above treatment were further confirmed by both real-time RT-PCR and the ECA assay.

Conclusion

Taken together, our results suggested that melatonin might synergize with pro-inflammatory cytokines to modulate the asthma airway inflammation through promoting the expression of chemotaxins in lung epithelial cell.     

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Background

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.

Methods

As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.

Results

Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.

Conclusion

Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.