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.     

Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI)

Background

The role of serum anti-Müllerian hormone (AMH) as predictor of in-vitro fertilization outcomes has been much debated. The aim of the present study is to investigate the practicability of combining serum AMH level with biological age as a simple screening method for counseling IVF candidates of advanced reproductive age with potential poor outcomes prior to treatment initiation.

Methods

A total of 1,538 reference patients and 116 infertile patients aged greater than or equal to 40 years enrolled in IVF/ICSI cycles were recruited in this retrospective analysis. A reference chart of the age-related distribution of serum AMH level for Asian population was first created. IVF/ICSI patients aged greater than or equal to 40 years were then divided into three groups according to the low, middle and high tertiles the serum AMH tertiles derived from the reference population of matching age. The cycle outcomes were analyzed and compared among each individual group.

Results

For reference subjects aged greater than or equal to 40 years, the serum AMH of the low, middle and high tertiles were equal or lesser than 0.48, 0.49-1.22 and equal or greater than 1.23 ng/mL respectively. IVF/ICSI patients aged greater than or equal to 40 years with AMH levels in the low tertile had the highest cycle cancellation rate (47.6%) with zero clinical pregnancy. The nadir AMH level that has achieved live birth was 0.56 ng/mL, which was equivalent to the 36.4th percentile of AMH level from the age-matched reference group. The optimum cut-off levels of AMH for the prediction of nonpregnancy and cycle cancellation were 1.05 and 0.68 ng/mL, respectively.

Conclusions

Two criteria: (1) age greater than or equal to 40 years and (2) serum AMH level in the lowest tertile (equal or lesser than 33.3rd percentile) of the matching age group, may be used as markers of futility for counseling IVF/ICSI candidates.     

Relationship between onion thrips (Thrips tabaci) and Stemphylium vesicarium in the development of Stemphylium leaf blight in onion

Stemphylium leaf blight caused by Stemphylium vesicarium and onion thrips (Thrips tabaci) are two common causes of leaf damage in onion production. Onion thrips is known to interact synergistically with pathogens to exacerbate plant disease. However, the potential relationship between onion thrips and Stemphylium leaf blight is unknown. In a series of controlled laboratory and field trials, the relationship between thrips feeding and movement on the development and severity of Stemphylium leaf blight were examined. In laboratory assays, onions (“Avalon” and “Ailsa Craig”) with varying levels of thrips feeding damage were inoculated with S. vesicarium. Pathogen colonisation and leaf dieback were measured after 2 weeks. In pathogen transfer assays, thrips were exposed to S. vesicarium conidia, transferred to onion and leaf disease development was monitored. In field trials, insecticide use was examined as a potential indirect means to reduce Stemphylium leaf blight disease and pathogen colonisation by reducing thrips damage. Results from laboratory trials revealed that a reduction in thrips feeding decreased S. vesicarium colonisation of onion leaves by 2.3–2.9 times, and decreased leaf dieback by 40–50%. Additionally, onion thrips were capable of transferring S. vesicarium conidia to onion plants (albeit at a low frequency of 2–14% of plants inoculated). In field trials, the symptoms and colonisation of Stemphylium leaf blight were reduced by 27 and 17%, respectively with the use of insecticide to control thrips. These results suggest that onion thrips may play a significant role in the development of Stemphylium leaf blight, and thrips control may reduce disease in commercial onion fields.     

DnaK prevents human insulin amyloid fiber formation on hydrophobic surfaces

We have developed a multiwell-based protein aggregation assay to study the kinetics of insulin adsorption and aggregation on hydrophobic surfaces and to investigate the molecular mechanisms involved. Protein-surface interaction progresses in two phases: (1) a lag phase during which proteins adsorb and prefibrillar aggregates form on the material surface and (2) a growth phase during which amyloid fibers form and then are progressively released into solution. We studied the effect of three bacterial chaperones, DnaK, DnaJ, and ClpB, on insulin aggregation kinetics. In the presence of ATP, the simultaneous presence of DnaK, DnaJ, and ClpB allows good protection of insulin against aggregation. In the absence of ATP, DnaK alone is able to prevent insulin aggregation. Furthermore, DnaK binds to insulin adsorbed on hydrophobic surfaces. This process is slowed in the presence of ATP and can be enhanced by the cochaperone DnaJ. The peptide LVEALYL, derived from the insulin B chain, is known to promote fast aggregation in a concentration- and pH-dependent manner in solution. We show that it also shortens the lag phase for insulin aggregation on hydrophobic surfaces. As this peptide is also a known DnaK substrate, our data indicate that the peptide and the chaperone might compete for a common site during the process of insulin aggregation on hydrophobic surfaces.     

Temperature and precipitation affect seasonal patterns of dispersing tobacco thrips, Frankliniella fusca, and onion thrips, Thrips tabaci (Thysanoptera: Thripidae) caught on sticky traps

Effects of temperature and precipitation on the temporal patterns of dispersing tobacco thrips, Frankliniella fusca, and onion thrips, Thrips tabaci, caught on yellow sticky traps were estimated in central and eastern North Carolina and eastern Virginia from 1997 through 2001. The impact that these environmental factors had on numbers of F. fusca and T. tabaci caught on sticky traps during April and May was determined using stepwise regression analysis of 43 and 38 site-years of aerial trapping data from 21 and 18 different field locations, respectively. The independent variables used in the regression models included degree-days, total precipitation, and the number of days in which precipitation occurred during January through May. Each variable was significant in explaining variation for both thrips species and, in all models, degree-days was the single best explanatory variable. Precipitation had a comparatively greater effect on T. tabaci than F. fusca. The numbers of F. fusca and T. tabaci captured in flight were positively related to degree-days and the number of days with precipitation but negatively related to total precipitation. Combined in a single model, degree-days, total precipitation, and the number of days with precipitation explained 70 and 55% of the total variation in the number of F. fusca captured from 1 April through 10 May and from 1 April through 31 May, respectively. Regarding T. tabaci flights, degree-days, total precipitation, and the number of days with precipitation collectively explained 57 and 63% of the total variation in the number captured from 1 April through 10 May and from 1 April through 31 May, respectively.     

Establishment of multicolored Asian lady beetle in Eastern North Carolina: Seasonal abundance and crop exploitation within an agricultural landscape

Seasonal abundance and cropexploitation of the multicolored Asian ladybeetle, Harmonia axyridis (Pallas)(Coccinellidae: Coleoptera), were identifiedduring its initial establishment in the easternNorth Carolina agroecosystem. Densities ofH. axyridis adults and larvae werecompared with densities of previouslyestablished predaceous coccinellids in wheat,potato, corn and soybean, which are thepredominant crops in this region. One-hundred-fifty whole plant samples werevisually inspected for coccinellid adults andlarvae in each crop on six farms every 7 to 14days. H. axyridis adults colonizedwheat, potato and corn, but reproduced only inwheat and potato. Soybean fields were notcolonized. The presence of H. axyridisin a crop was typically associated with anabundance of aphids, with the exception ofpotato, and no aphids were encountered insoybean. In addition to H. axyridis,four other lady beetle species, Coccinellaseptempunctata L., Coleomegilla maculata(DeGeer), Hippodamia convergens (Guerin)and Cycloneda munda L., were encounteredin the landscape. In wheat, potato and corn,densities of H. axyridis adults averagedthroughout the 1995 and 1996 seasons wereseven, ten and 28 times lower, respectively,than the season average density of the leastabundant species of previously established ladybeetle, whereas these densities averaged 82, 42and 356 times lower, respectively, than theaverage density of the most abundantestablished coccinellid species. H.axyridis is commonly encountered in theeastern North Carolina agricultural landscape,but its impact on the existing coccinellidassemblage cannot yet be determined. Theresults presented provide a baseline againstwhich the results of future studies can becompared to determine if H. axyridis isdisplacing established species.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

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.