Low back pain is a common and debilitating disorder. Current evidence implicates intervertebral disc (IVD) degeneration and herniation as major causes, although the pathogenesis is poorly understood. ...While several cytokines have been implicated in the process of IVD degeneration and herniation, investigations have predominately focused on Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF alpha ). However, to date no studies have investigated the expression of these cytokines simultaneously in IVD degeneration or herniation, or determined which may be the predominant cytokine associated with these disease states. Using quantitative real time PCR and immunohistochemistry we investigated gene and protein expression for IL-1 beta , TNF alpha and their receptors in non-degenerate, degenerate and herniated human IVDs. IL-1 beta gene expression was observed in a greater proportion of IVDs than TNF alpha (79% versus 59%). Degenerate and herniated IVDs displayed higher levels of both cytokines than non-degenerate IVDs, although in degenerate IVDs higher levels of IL-1 beta gene expression (1,300 copies/100 ng cDNA) were observed compared to those of TNF alpha (250 copies of TNF alpha /100 ng cDNA). Degenerate IVDs showed ten-fold higher IL-1 receptor gene expression compared to non-degenerate IVDs. In addition, 80% of degenerate IVD cells displayed IL-1 receptor immunopositivity compared to only 30% of cells in non-degenerate IVDs. However, no increase in TNF receptor I gene or protein expression was observed in degenerate or herniated IVDs compared to non-degenerate IVDs. We have demonstrated that although both cytokines are produced by human IVD cells, IL-1 beta is expressed at higher levels and in more IVDs, particularly in more degenerate IVDs (grades 4 to 12). Importantly, this study has highlighted an increase in gene and protein production for the IL-1 receptor type I but not the TNF receptor type I in degenerate IVDs. The data thus suggest that although both cytokines may be involved in the pathogenesis of IVD degeneration, IL-1 may have a more significant role than TNF alpha , and thus may be a better target for therapeutic intervention.
In this study, we investigated the hypotheses that in human intervertebral disc (IVD) degeneration there is local production of the cytokine IL-1, and that this locally produced cytokine can induce ...the cellular and matrix changes of IVD degeneration. Immunohistochemistry was used to localize five members of the IL-1 family (IL-1alpha, IL-1beta, IL-1Ra (IL-1 receptor antagonist), IL-1RI (IL-1 receptor, type I), and ICE (IL-1beta-converting enzyme)) in non-degenerate and degenerate human IVDs. In addition, cells derived from non-degenerate and degenerate human IVDs were challenged with IL-1 agonists and the response was investigated using real-time PCR for a number of matrix-degrading enzymes, matrix proteins, and members of the IL-1 family. This study has shown that native disc cells from non-degenerate and degenerate discs produced the IL-1 agonists, antagonist, the active receptor, and IL-1beta-converting enzyme. In addition, immunopositivity for these proteins, with the exception of IL-1Ra, increased with severity of degeneration. We have also shown that IL-1 treatment of human IVD cells resulted in increased gene expression for the matrix-degrading enzymes (MMP 3 (matrix metalloproteinase 3), MMP 13 (matrix metalloproteinase 13), and ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs)) and a decrease in the gene expression for matrix genes (aggrecan, collagen II, collagen I, and SOX6). In conclusion we have shown that IL-1 is produced in the degenerate IVD. It is synthesized by native disc cells, and treatment of human disc cells with IL-1 induces an imbalance between catabolic and anabolic events, responses that represent the changes seen during disc degeneration. Therefore, inhibiting IL-1 could be an important therapeutic target for preventing and reversing disc degeneration.
Aims
This study is the first to systematically document histological features of fractures of known age in infants (≦12 months). It has been used to develop a tabulated database specifically to guide ...histopathologists to age fractures in children considered to have suffered accidental or non‐accidental injury (NAI). Currently in the United Kingdom there are insufficient pathologists with experience in histological ageing of fractures to meet the medicolegal need for this examination. This study provides a practical tool that will allow those skilled paediatric and forensic pathologists currently involved in assessing infants for evidence of accidental or non‐accidental injury a basis for extending their assessment into this area of unmet need.
Methods and results
One hundred and sixty‐nine fractures of known age at death were obtained from 52 anonymised infants over a period of 32 years (1985–2016 inclusive). Sections stained using haematoxylin and eosin (H&E) and Martius scarlet blue (MSB) were used to identify specific histological features and to relate them to fracture age. In 1999 the data were entered into a tabulated database for fractures accumulated between from 1985 to 1998 inclusive. Thereafter cases were added, and at 2‐yearly intervals the accumulated data were audited against the previous database and adjustments made.
Conclusions
This paper describes the final data set from the 2017 audit. The study was terminated at the end of 2016, as there had been no material changes in the data set for three consecutive audits.
The nucleus pulposus (NP) of the intervertebral disc (IVD) demonstrates substantial changes in cell and matrix composition with both ageing and degeneration. While recent transcriptomic profiling ...studies have helped define human NP cell phenotype, it remains unclear how expression of these markers is influenced by ageing or degeneration. Furthermore, cells of the NP are thought to derive from the notochord, although adult NP lacks identifiable notochordal (NC) cells. This study aimed to confirm expression of previously identified NP and NC marker genes in adult human NP cells from a range of ages and degenerate states. Importantly, using gene expression analysis (N = 60) and immunohistochemistry (N = 56) the study demonstrates expression of NP markers FoxF1, Pax-1, keratin-8/18, carbonic anhydrase-12, and NC markers brachyury, galectin-3 and CD24 in cells of the NP irrespective of age or degeneration. Our immunohistochemical data, combined with flow cytometry (N = 5) which identified a small number of CA12
Gal3
T
CD24
cells, suggests the possible presence of a sub-population of cells with an NC-like phenotype in adult NP tissue. These findings suggest that the NP contains a heterogeneous population of cells, which may possess varied phenotypic and functional profiles and thus warrant further investigation to improve our understanding of IVD homeostasis and repair.
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.
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-1beta or TNFalpha 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.
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-1beta led to significant increases in NGF and BDNF gene expression (P < 0.05). Treatment with TNFalpha was associated with an upregulation of substance P expression only.
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-1beta and modulation of substance P expression by TNFalpha, 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.
The circadian clock plays a crucial role in regulating physiology and is important for maintaining immune homeostasis and responses to inflammatory stimuli. Inflammatory arthritis often shows diurnal ...variation in disease symptoms and disease markers, and it is now established that cellular clocks regulate joint inflammation. The clock gene Bmal1 is critical for maintenance of 24-h rhythms and plays a key role in regulating immune responses, as well as in aging-related processes. Fibroblast-like synoviocytes (FLS) are circadian rhythmic joint mesenchymal cells which are important for maintenance of joint health and play a crucial role in the development of inflammatory arthritis. The aim of this study was to investigate the importance of the joint mesenchymal cell circadian clock in health and disease.
Mice were generated which lack Bmal1 in Col6a1-expressing cells, targeting mesenchymal cells in the ankle joints. Joints of these animals were assessed by X-ray imaging, whole-mount staining and histology, and the composition of the synovium was assessed by flow cytometry. Arthritis was induced using collagen antibodies.
Bmal1 deletion in joint mesenchymal cells rendered the FLS and articular cartilage cells arrhythmic. Targeted mice exhibited significant changes in the architecture of the joints, including chondroid metaplasia (suggesting a switch of connective tissue stem cells towards a chondroid phenotype), reductions in resident synovial macrophages and changes in the basal pro-inflammatory activity of FLS. Loss of Bmal1 in FLS rendered these resident immune cells more pro-inflammatory in response to challenge, leading to increased paw swelling, localised infiltration of mononuclear cells and enhanced cytokine production in a model of arthritis.
This study demonstrates the importance of Bmal1 in joint mesenchymal cells in regulating FLS and chondrocyte development. Additionally, we have identified a role for this core clock component for restraining local responses to inflammation and highlight a role for the circadian clock in regulating inflammatory arthritis.
Innervation of nociceptive nerve fibres into the normally aneural nucleus pulposus (NP) of the intervertebral disc (IVD) occurs during degeneration resulting in discogenic back pain. The ...neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), which are associated with stimulation of axonal outgrowth and nociception by neuronal cells, are both expressed by NP cells, with BDNF levels increasing with disease severity. However the mechanism of interaction between human NP cells and neural cells has yet to be fully elucidated. Therefore the aim of this study was to determine whether non-degenerate or degenerate human NP cells inhibit or stimulate neural outgrowth and whether any outgrowth is mediated by NGF or BDNF. Human NP cells from non-degenerate and degenerate IVD were cultured in alginate beads then co-cultured for 48 hours with human SH-SY5Y neuroblastoma cells. Co-culture of non-degenerate NP cells with neural cells resulted in both an inhibition of neurite outgrowth and reduction in percentage of neurite expressing cells. Conversely co-culture with degenerate NP cells resulted in an increase in both neurite length and percentage of neurite expressing cells. Addition of anti-NGF to the co-culture with degenerate cells resulted in a decrease in percentage of neurite expressing cells, while addition of anti-BDNF resulted in a decrease in both neurite length and percentage of neurite expressing cells. Our findings show that while non-degenerate NP cells are capable of inhibiting neurite outgrowth from human neural cells, degenerate NP cells stimulate outgrowth. Neurotrophin blocking studies demonstrated that both NGF and BDNF, secreted by degenerate NP cells, may play a role in this stimulation with BDNF potentially playing the predominant role. These findings suggest that NP cells are capable of regulating nerve ingrowth and that neoinnervation occurring during IVD degeneration may be stimulated by the NP cells themselves.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) on protease and catabolic cytokine and receptor gene expression in ...normal and degenerate human nucleus pulposus cells in alginate culture.
Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-alpha or IL-1beta for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-alpha or IL-beta stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-alpha, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1alpha, IL-1beta, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra).
MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1beta than TNF-alpha. MMP-13 was upregulated by each cytokine to a similar extent. TNF-alpha and TNF-R2 expressions were upregulated by both TNF-alpha and IL-beta, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1beta and IL-1Ra expressions were significantly upregulated by TNF-alpha, whereas IL-1alpha and IL-1R1 were unchanged.
TNF-alpha does not induce MMP expression to the same degree as stimulation by IL-1beta, but it does act to upregulate IL-1beta expression as well as TNF-alpha and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-alpha may be an important initiating factor in matrix degeneration, IL-1beta plays a greater role in established pathological degradation.
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.
Microarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip(R) Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells.
Microarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed > or =100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells, although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells.
This study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles, and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separated NP and NC cell populations suggests that these two cell types may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount.
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