Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.     

High cholesterol inhibits tendon-related gene expressions in tendon-derived stem cells through reactive oxygen species-activated nuclear factor-κB signaling

Clinical studies have indicated that increased serum cholesterol levels raised the risk of tendinopathy in hypercholesterolemia, but the effect of cholesterol on tendon-derived stem cells (TDSCs) and its underlying mechanism have not been studied. The purpose of this study is to investigate the association between cholesterol and tendinopathy in vitro and in vivo, and its underlying molecular mechanism as well. In TDSCs, the effect of cholesterol was assessed by quantitative polymerase chain reaction, western blot analysis, and immunofluorescence staining. Intracellular levels of reactive oxygen species (ROS) was detected, using flow cytometry. The link between nuclear factor (NF)-κB signaling and the effect of cholesterol was evaluated using a representative IκB kinase (IKK) inhibitor, BAY 11-7082. In addition, Achilles tendons from apolipoprotein E mice fed with a high-fat diet were histologically assessed using hematoxylin and eosin staining and immunohistochemistry. We found that high cholesterol apparently lowered the expression of tendon cell markers (collagen 1, scleraxis, tenomodulin), and elevated ROS levels via the NF-κB pathway both in vitro and in vivo. The ROS scavenger N-acetylcysteine (NAC) and BAY 11-7082 reversed the inhibiting effect of cholesterol on the tendon-related gene expressions of TDSCs. Moreover, NAC blocked cholesterol-induced phosphorylation of IκBα and p65. Significant histological alternation in vivo was shown in Achilles tendon in the hypercholesterolemic group. These results indicated that high cholesterol may inhibit the tendon-related gene expressions in TDSCs via ROS-activated NF-кB signaling, implying pathogenesis of tendinopathy in hypercholesterolemia and suggesting a new mechanism underlying hypercholesterolemia-induced tendinopathy.     

Uphill running improves rat Achilles tendon tissue mechanical properties and alters gene expression without inducing pathological changes

Overuse Achilles tendinopathy is a common and challenging problem in sports medicine. Little is known about the etiology of this disorder, and the development of a good animal model for overuse tendinopathy is essential for advancing insight into the disease mechanisms. Our aim was to test a previously proposed rat model for Achilles tendon overuse. Ten adult male Sprague-Dawley rats ran on a treadmill with 10° incline, 1 h/day, 5 days/wk (17-20 m/min) for 12 wk and were compared with 12 control rats. Histological, mechanical, and gene-expression changes were measured on the Achilles tendons after the intervention, and local tendon glucose-uptake was measured before and after the intervention with positron emission tomography. No differences were detected between runners and controls in tissue histology or in glucose uptake, indicating that tendon pathology was not induced. Greater tendon tissue modulus (P < 0.005) and failure stress/body weight (P < 0.02) in runners compared with controls further supported that tendons successfully adapted to uphill running. Several genes of interest were regulated after 12 wk of running. Expression of collagen III and insulin-like growth factor I was increased, while collagen I was unchanged, and decreases were seen in noncollagen matrix components (fibromodulin and biglycan), matrix degrading enzymes, transforming growth factor-β1, and connective tissue growth factor. In conclusion, the tested model could not be validated as a model for Achilles tendinopathy, as the rats were able to adapt to 12 wk of uphill running without any signs of tendinopathy. Improved mechanical properties were observed, as well as changes in gene-expression that were distinctly different from what is seen in tendinopathy and in response to short-term tendon loading.     

Gap junctions in IL-1β-mediated cell survival response to strain

Mechanical stimuli play important roles in proliferation and differentiation of connective tissue cells, and development and homeostatic maintenance of tissues. However, excessive mechanical loading to a tissue can injure cells and disrupt the matrix, as occurs in tendinopathy. Tendinopathy is a common clinical problem in athletes and in many occupational settings due to overuse of the tendon. Moreover, interleukin (IL)-1β is generally considered to be a "bad" cytokine, activating NF-κb and cell death and inducing matrix metalloproteinase (MMPs 1, 2, 3) expression and matrix destruction. However, activated NF-κB can also drive a cell survival pathway. We have reported that cyclic strain induced tenocyte death in three-dimensional (3D) cultures, and IL-1β could promote cell survival under strain. Therefore, it was hypothesized that 1) cyclic strain could induce cell death in tenocytes as observed in pathologic tendons in vivo; 2) a gene expression profile indicative of tendinopathy could be identified; and 3) low-dose IL-1β could protect cells from strain-induced, tendinopathy-like changes. Human tenocytes were cultured in 3D type I collagen hydrogels and subjected to 3.5% elongation at 1 Hz for 1 h/day for up to 5 days with or without IL-1β. Real-time RT-PCR data showed that cyclic strain regulated the expression of tendinopathy marker genes in a manner similar to that found in pathological tendons from patients and that addition of IL-1β reversed the gene expression changes to control levels. Results of further studies showed that IL-1β may modulate cell survival through upregulating the expression of connexin 43, which is involved in the modulation of cell death/survival in a variety of cells and tissues. The elucidation of the mechanisms underlying strain-induced cell death and recovery from strain injury will facilitate our understanding of the pathogenesis of tendinopathy and may lead to the discovery of new molecular targets for early diagnosis and treatment of tendinopathy.     

Creating an Animal Model of Tendinopathy by Inducing Chondrogenic Differentiation with Kartogenin

Previous animal studies have shown that long term rat treadmill running induces over-use tendinopathy, which manifests as proteoglycan accumulation and chondrocytes-like cells within the affected tendons. Creating this animal model of tendinopathy by long term treadmill running is however time-consuming, costly and may vary among animals. In this study, we used a new approach to develop an animal model of tendinopathy using kartogenin (KGN), a bio-compound that can stimulate endogenous stem/progenitor cells to differentiate into chondrocytes. KGN-beads were fabricated and implanted into rat Achilles tendons. Five weeks after implantation, chondrocytes and proteoglycan accumulation were found at the KGN implanted site. Vascularity as well as disorganization in collagen fibers were also present in the same site along with increased expression of the chondrocyte specific marker, collagen type II (Col. II). In vitro studies confirmed that KGN was released continuously from KGN-alginate in vivo beads and induced chondrogenic differentiation of tendon stem/progenitor cells (TSCs) suggesting that chondrogenesis after KGN-bead implantation into the rat tendons is likely due to the aberrant differentiation of TSCs into chondrocytes. Taken together, our results showed that KGN-alginate beads can be used to create a rat model of tendinopathy, which, at least in part, reproduces the features of over-use tendinopathy model created by long term treadmill running. This model is mechanistic (stem cell differentiation), highly reproducible and precise in creating localized tendinopathic lesions. It is expected that this model will be useful to evaluate the effects of various topical treatments such as NSAIDs and platelet-rich plasma (PRP) for the treatment of tendinopathy.     

Confocal arthroscopy-based patient-specific constitutive models of cartilaginous tissues--I: development of a microstructural model

Current development of a laser scanning confocal arthroscope within our school will enable 3D microscopic imaging of joint tissues in vivo. Such an instrument could be useful, for example, in assessing the microstructural condition of the living tissues without physical biopsy. It is envisaged also that linked to a suitable microstructural constitutive formulation, such imaging could allow non-invasive patient-specific estimation of tissue mechanical performance. Such a procedure could have applications in surgical planning and simulation, and assessment of engineered tissue replacements, where tissue biopsy is unacceptable. In this first of two papers the development of a suitable constitutive framework for generating such estimates is reported. A microstructure-based constitutive formulation for cartilaginous tissues is presented. The model extends existing fibre composite-type models and accounts for strain-rate sensitivity of the tissue mechanical response through incorporation of a viscoelastic fibre phase. Importantly, the model is constructed so as to allow direct incorporation of structural data from confocal images. A finite element implementation of the formulation suitable for incorporation within commercial codes is also presented.     

Hypoxia and inflammatory bowel disease

Inflammatory bowel disease (IBD) is a general term to describe inflammatory diseases of the gastrointestinal tract such as Crohn's disease and ulcerative colitis. IBD affects approximately 1 in 200 individuals and exerts a significant health and quality of life burden on patients. Surgical intervention can be curative in ulcerative colitis but there is currently no cure for Crohn's disease. Since this is the case, and the fact that patients are often diagnosed at a young age, IBD exerts a significant financial burden on the health care system, and society as a whole.The underlying pathology of IBD is complex and involves a combination of genetic, environmental and microbial factors. Regardless of the underlying causes of the condition, this disease is universally characterized by disruption to the protective epithelial barrier separating the intestinal lumen above from the mucosal immune system below. Once this barrier becomes compromised a sequence of events ensues, that can occur in repetitive cycles to ensure long-term and serious damage to the gut.The role of hypoxia and hypoxia-dependent signalling pathways are increasingly appreciated to play a role in the physiology and pathophysiology of the intestine. The intestinal epithelium normally exists in a state of physiological hypoxia, with additional tissue hypoxia a feature of active inflammatory disease. Furthermore, recent pre-clinical animal studies have clearly supported the rationale for pharmacologically manipulating the oxygen-sensitive hypoxia-inducible factor (HIF) pathway in models of IBD. Thus, this review will discuss the contribution of hypoxia sensitive pathways in the pathology of IBD. Finally we will discuss the emerging evidence for manipulation of hypoxia-sensitive pathways in the treatment of IBD.     

Reasoning in uncertainties. An analysis of five strategies and their suitability in pathology

In reasoning systems, uncertainty plays a crucial part, especially for those fields in which judgements are essential, as in pathology. Uncertainty has several aspects, such as prevalence of diseases, occurrence of findings and the sensitivity and predictive value of findings. For the functioning of a reasoning system, two aspects are crucial: (1) the internal representation of the uncertainty and (2) the way in which the uncertainty is propagated in the reasoning process when combining formal statements. Five well-known reasoning strategies (Bayes' probability theory, MYCIN's certainty factor model, fuzzy set theory, the theory of Dempster-Shafer and Pathfinder's scoring mechanism) are compared, with particular attention to: (1) Under what conditions will the model function? In particular, what information is to be specified a priori to the system? (2) Can the different aspects of uncertainty be dealt with as separate entities? (3) How are unknown uncertainties dealt with? (4) How is evidence in favor of a hypothesis combined with evidence against it? (5) How does the model treat the simultaneous occurrence of more than one disorder, that is, how does the model support reasoning with compound hypotheses? It is preliminarily concluded that the different aspects of uncertainty are expressed as separate entities only in Pathfinder and probability theory. Hence, the other models do not accurately represent uncertain knowledge. Also, such theoretically attractive models as the Bayes, MYCIN and Dempster-Shafer theory can only function properly under the tight condition of mutual exclusiveness of hypotheses, which is not always suited for broader areas of pathology. They may, however, be suited for smaller areas, with a limited number of defined diseases and a limited number of features. All models but the Bayes model lack a predictable performance since there is no (or only a partial) underlying theory to guarantee minimization of the overall error.     

Comparative Analysis of Hepatic CD14 Expression between Two Different Endotoxin Shock Model Mice: Relation between Hepatic Injury and CD14 Expression

CD14 is a glycoprotein that recognizes gram-negative bacterial lipopolysaccharide (LPS) and exists in both membrane-bound and soluble forms. Infectious and/or inflammatory diseases induce CD14 expression, which may be involved in the pathology of endotoxin shock. We previously found that the expression of CD14 protein differs among the endotoxin shock models used, although the reasons for these differences are unclear. We hypothesized that the differences in CD14 expression might be due to liver injury, because the hepatic tissue produces CD14 protein. We investigated CD14 expression in the plasma and liver in the carrageenan (CAR)-primed and D-galN-primed mouse models of endotoxin shock. Our results showed that severe liver injury was not induced in CAR-primed endotoxin shock model mice. In this CAR-primed model, the higher mRNA and protein expression of CD14 was observed in the liver, especially in the interlobular bile duct in contrast to D-galN-primed-endotoxin shock model mice. Our findings indicated that the molecular mechanism(s) underlying septic shock in CAR-primed and D-galN-primed endotoxin shock models are quite different. Because CD14 expression is correlated with clinical observations, the CAR-primed endotoxin shock model might be useful for studying the functions of CD14 during septic shock in vivo.     

HIV-specific probabilistic models of protein evolution

Comparative sequence analyses, including such fundamental bioinformatics techniques as similarity searching, sequence alignment and phylogenetic inference, have become a mainstay for researchers studying type 1 Human Immunodeficiency Virus (HIV-1) genome structure and evolution. Implicit in comparative analyses is an underlying model of evolution, and the chosen model can significantly affect the results. In general, evolutionary models describe the probabilities of replacing one amino acid character with another over a period of time. Most widely used evolutionary models for protein sequences have been derived from curated alignments of hundreds of proteins, usually based on mammalian genomes. It is unclear to what extent these empirical models are generalizable to a very different organism, such as HIV-1-the most extensively sequenced organism in existence. We developed a maximum likelihood model fitting procedure to a collection of HIV-1 alignments sampled from different viral genes, and inferred two empirical substitution models, suitable for describing between-and within-host evolution. Our procedure pools the information from multiple sequence alignments, and provided software implementation can be run efficiently in parallel on a computer cluster. We describe how the inferred substitution models can be used to generate scoring matrices suitable for alignment and similarity searches. Our models had a consistently superior fit relative to the best existing models and to parameter-rich data-driven models when benchmarked on independent HIV-1 alignments, demonstrating evolutionary biases in amino-acid substitution that are unique to HIV, and that are not captured by the existing models. The scoring matrices derived from the models showed a marked difference from common amino-acid scoring matrices. The use of an appropriate evolutionary model recovered a known viral transmission history, whereas a poorly chosen model introduced phylogenetic error. We argue that our model derivation procedure is immediately applicable to other organisms with extensive sequence data available, such as Hepatitis C and Influenza A viruses.     

Mutagenicity testing with transgenic mice. Part II: Comparison with the mouse spot test

The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue(R) mouse), and the lacZ model (commercially available as the Mutatrade mark Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo.     

Temporal profile of amyloid-beta (Abeta) oligomerization in an in vivo model of Alzheimer disease. A link between Abeta and tau pathology

Accumulation of amyloid-beta (Abeta) is one of the earliest molecular events in Alzheimer disease (AD), whereas tau pathology is thought to be a later downstream event. It is now well established that Abeta exists as monomers, oligomers, and fibrils. To study the temporal profile of Abeta oligomer formation in vivo and to determine their interaction with tau pathology, we used the 3xTg-AD mice, which develop a progressive accumulation of plaques and tangles and cognitive impairments. We show that SDS-resistant Abeta oligomers accumulate in an age-dependent fashion, and we present evidence to show that oligomerization of Abeta appears to first occur intraneuronally. Finally, we show that a single intrahippocampal injection of a specific oligomeric antibody is sufficient to clear Abeta pathology, and more importantly, tau pathology. Therefore, Abeta oligomers may play a role in the induction of tau pathology, making the interference of Abeta oligomerization a valid therapeutic target.     

Triceps surae activation is altered in male runners with Achilles tendinopathy

Achilles tendinopathy is a common injury in running sports however the exact etiology of Achilles injury is still unclear. In recent years, altered neuromotor recruitment patterns of the triceps surae have been hypothesized to create differential intra-tendinous loads leading to pathology; however, this hypothesis has not been investigated. Further, the effect foot orthoses may have on neuromotor recruitment of the triceps surae in Achilles tendinopathy has not been investigated. Methods: The electromyographic activity of the triceps surae was recorded during an over-ground running task. Fifteen Achilles injured participants and 19 asymptomatic controls were assessed in a footwear only condition. The Achilles injured participants were also assessed running in a pre-fabricated foot orthoses. Results: In Achilles injured participants, there was a significant difference between soleus and lateral gastrocnemius offset times during running compared to the asymptomatic controls (p < 0.05). There were no significant differences in triceps surae muscle activity between the footwear only and footwear and orthoses condition in the Achilles injured participants. Conclusions: The finding that triceps surae activity is altered in participants with Achilles tendinopathy may have clinical importance as it suggests that intra-tendinous loads are altered which may contribute to pathological changes. Further, foot orthoses have no immediate effect on the neuromotor control of the triceps surae.     

Models of dementia: an introductory overview

The analysis of the molecular development of AD (Alzheimer's disease) is technically challenging, due to the chronic nature of the disease, the lack of early and definitive clinical diagnosis, and the fact that the abnormal molecular pathology occurs in the brain. Therefore appropriate animal models of AD are essential if we are to dissect the processes leading to molecular pathology, and ultimately to test the efficacy of potential therapies before clinical studies. Unfortunately, there is controversy over the benefits of the available models, the only consensus of opinion being that no perfect model currently exists. The investigation of animal models is extremely costly and time-consuming, therefore researchers tend to focus on one or two models. For scientists entering the AD research field, it can be difficult to identify the most appropriate model for their needs. Therefore the Models of Dementia: the Good, the Bad and the Future Biochemical Society Focused Meeting provided a platform for discussion and debate on the use and limitations of current models, the most appropriate methods for their characterization and identification of the most pressing needs of the field in general.     

自发性树鼩乳腺肿瘤的特性(英文)

乳腺癌是严重危害女性健康的常见恶性肿瘤,建立合适的乳腺癌动物模型对于研究人类乳腺癌的生物学机制及发展新的防治方法至关重要。相对于常用的啮齿类动物,树鼩(Tupaia belangeri chinensis,tree shrew)因在进化层次上更接近于人类而可用于建立更适合的乳腺癌模型。该文详细了介绍一例树鼩自发性乳头状良性乳腺肿瘤。免疫组化结果显示该例肿瘤孕激素受体阳性且Ki-67阳性细胞比例显著增加;而活化的Caspase3阳性细胞比例较低;且肿瘤的形态和病理与人导管内乳头状肿瘤非常接近。提示利用树鼩建立乳腺肿瘤模型的可行性。     

An Analytical Model for the ‘Large, Fluctuating Synaptic Conductance State’ Typical of Neocortical Neurons In Vivo

A model of in vivo-like neocortical activity is studied analytically in relation to experimental data and other models in order to understand the essential mechanisms underlying such activity. The model consists of a network of sparsely connected excitatory and inhibitory integrate-and-fire (IF) neurons with conductance-based synapses. It is shown that the model produces values for five quantities characterizing in vivo activity that are in agreement with both experimental ranges and a computer-simulated Hodgkin-Huxley model adapted from the literature (Destexhe et al. (2001) Neurosci. 107(1): 13-24). The analytical model builds on a study by Brunel (2000) (J. Comput. Neurosci. 8: 183-208), which used IF neurons with current-based synapses, and therefore does not account for the full range of experimental data. The present results suggest that the essential mechanism required to explain a range of data on in vivo neocortical activity is the conductance-based synapse and that the particular model of spike initiation used is not crucial. Thus the IF model with conductance-based synapses may provide a basis for the analytical study of the "large, fluctuating synaptic conductance state" typical of neocortical neurons in vivo.     

Understanding cellular and molecular mechanisms of pathogenesis of diabetic tendinopathy

There is accumulating evidence of an increased incidence of tendon disorders in people with diabetes mellitus. Diabetic tendinopathy is an important cause of chronic pain, restricted activity, and even tendon rupture in individuals. Tenocytes and tendon stem/progenitor cells (TSPCs) are the dominant cellular components associated with tendon homeostasis, maintenance, remodeling, and repair. Some previous studies have shown alterations in tenocytes and TSPCs in high glucose or diabetic conditions that might cause structural and functional variations in diabetic tendons and even accelerate the development and progression of diabetic tendinopathy. In this review, the biomechanical properties and histopathological changes in diabetic tendons are described. Then, the cellular and molecular alterations in both tenocytes and TSPCs are summarized, and the underlying mechanisms involved are also analyzed. A better understanding of the underlying cellular and molecular pathogenesis of diabetic tendinopathy would provide new insight for the exploration and development of effective therapeutics.     

Developing simulations to reproduce in vivo fluoroscopy kinematics in total knee replacement patients

For clinically predictive testing and design-phase evaluation of prospective total knee replacement (TKR) implants, devices should ideally be evaluated under physiological loading conditions which incorporate population-level variability. A challenge exists for experimental and computational researchers in determining appropriate loading conditions for wear and kinematic knee simulators which reflect in vivo joint loading conditions. There is a great deal of kinematic data available from fluoroscopy studies. The purpose of this work was to develop computational methods to derive anterior–posterior (A–P) and internal–external (I–E) tibiofemoral (TF) joint loading conditions from in vivo kinematic data. Two computational models were developed, a simple TF model, and a more complex lower limb model. These models were driven through external loads applied to the tibia and femur in the TF model, and applied to the hip, ankle and muscles in the lower limb model. A custom feedback controller was integrated with the finite element environment and used to determine the external loads required to reproduce target kinematics at the TF joint. The computational platform was evaluated using in vivo kinematic data from four fluoroscopy patients, and reproduced in vivo A–P and I–E motions and compressive force with a root-mean-square (RMS) accuracy of less than 1 mm, 0.1°, and 40 N in the TF model and in vivo A–P and I–E motions, TF flexion, and compressive loads with a RMS accuracy of less than 1 mm, 0.1°, 1.4°, and 48 N in the lower limb model. The external loading conditions derived from these models can ultimately be used to establish population variability in loading conditions, for eventual use in computational as well as experimental activity simulations.     

Symmetric competition as a general model for single-species adaptive dynamics

Adaptive dynamics is a widely used framework for modeling long-term evolution of continuous phenotypes. It is based on invasion fitness functions, which determine selection gradients and the canonical equation of adaptive dynamics. Even though the derivation of the adaptive dynamics from a given invasion fitness function is general and model-independent, the derivation of the invasion fitness function itself requires specification of an underlying ecological model. Therefore, evolutionary insights gained from adaptive dynamics models are generally model-dependent. Logistic models for symmetric, frequency-dependent competition are widely used in this context. Such models have the property that the selection gradients derived from them are gradients of scalar functions, which reflects a certain gradient property of the corresponding invasion fitness function. We show that any adaptive dynamics model that is based on an invasion fitness functions with this gradient property can be transformed into a generalized symmetric competition model. This provides a precise delineation of the generality of results derived from competition models. Roughly speaking, to understand the adaptive dynamics of the class of models satisfying a certain gradient condition, one only needs a complete understanding of the adaptive dynamics of symmetric, frequency-dependent competition. We show how this result can be applied to number of basic issues in evolutionary theory.