Colon cancer and obesity are two significant and related pathological states with multiple etiological factors. In this dissertation, it was hypothesized that tumor growth is accelerated in the altered state of obesity due to their resistance towards tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity. Physiologically elevated TNF-alpha in an obese state induces increased nuclear transcription factor-kB (NF-kB) activity, known to transcribe genes crucial to cell survival. Insulin resistance, oxidative stress, and a pro-inflammatory environment are few of the biological consequences of TNF-alpha and NF-kB pathway activation, and further contribute to disease progression. <br /><br /> Three major studies were conducted to investigate phenotypical changes in obesity associated tumors. Firstly, characteristics of the TNF-alpha resistant phenotype were preliminarily assessed by evaluating the effects of exogenous TNF-alpha treatment to HT-29 cells. Elevated levels of NF-kB in response to exogenous TNF-alpha gave an indication that this pathway is critical for cell survival. Furthermore, upregulation of TNF-alpha receptor 2 (TNFR2) suggested another strategy by which the cells were utilizing exogenous TNF-alpha for a survival advantage. Inhibition of NF-kB via St. John?s Wort treatment demonstrated that HT-29 cells may be sensitized towards TNF-alpha mediated cytotoxicity. <br /><br /> Zucker obese (Zk-Ob), Zucker lean (Zk-Ln), and Sprague Dawley (SD) animal models were used to assess tumor phenotype <em>in vivo</em>. Remarkable physiological differences between genotypes were observed. Zk-Ob rats had significantly higher body and organ weights as well as plasma TNF- alpha, insulin, leptin, and oxidative markers than Zk-Ln and SD animals. Tumor incidence and multiplicity were also notably higher in Zk-Ob rats. Protein analyses demonstrated increased levels of TNF-alpha, TNFR2, NF-kB, IkB kinase beta (IKKbeta), insulin receptor (IR), insulin like growth factor-I-receptor (IGF-IR), and mitogen activated protein kinase (MAPK) in Zk-Ob tumors than Zk-Ln counterparts. In all groups, tumors generally had higher protein expression than surrounding, normal appearing colonic mucosa. It is well known that these molecules are involved in signaling pathways that influence and co-operate with each other in rendering growth autonomy to tumor tissue. <br /><br /> A higher number of lesions in the distal than proximal colon in Zk-Ob rats was observed, supporting the emerging concept that genotype/physiological state of the host affects development and distribution of tumors. Thus, a third study was conducted to explore differences between distal and proximal tumor phenotype. Results demonstrated that expression of TNFR2, NF-kB, IR, IGF-IR, and MAPK p44 were significantly higher in distal than proximal tumors. This observation suggested that development of tumors in different regions of the colon varied under the same physiological conditions. Moreover, phenotype of distal tumors appeared to be upregulating survival pathways in comparison to proximal lesions, possibly explaining the higher tumor incidence in the distal colon. <br /><br /> Research documented in this thesis supported the hypothesis that the physiological status of the host intricately affects tumor phenotype. In particular, the TNF-alpha resistant phenotype was most prominent in Zk-Ob tumors, and appeared to be associated with upregulation of multiple signaling pathways cooperating towards tumorigenesis.

Skeletal muscle is defined to be atrophic in osteoporosis models and therefore is a potential target tissue for osteoporosis research. The aim of this longitudinal randomized controlled interdisciplinary study was to analyze the functional, histological, ultra-structral and molecular changes and the role of cachectic muscle atrophy inducer TNF-alpha in the skeletal muscles of the ovariectomized (OVX) rat model which mimics postmenopausal osteoporosis. Female Sprague-Dawley rats were randomly assigned to the control, the OVX and the OVX+10&amp / #956 / g/g/week TNF-alpha antagonist (Remicade) treated OVX-TNF groups. Maximum isometric and tetanic-twitch amplitudes were lower than the control group in the OVX group. Maximum isometric twitch amplitudes recovered in the fast-twitch extensor digitorum longus (EDL) muscles but not in the slow-twitch soleus muscles in the OVX-TNF group. The decrease in tetanic-twitch amplitudes recovered in the OVX-TNF group in both muscle types. Splitting and size variations of fibers, central nuclei and well-preserved overall ultrastructure were noted in the OVX and the OVX-TNF groups. Slow-twitch Type I fiber percentage, areas and diameters increased in EDL muscles of the OVX and the OVX-TNF group comparing to the control group. p65 and MyoD immune-labeling increased in OVX group whereas MyoD and C-Rel increased and p50 decreased in OVX-TNF group. Expressions of 61 genes and 42 unidentified transcripts were significantly different between the control, the OVX and the OVX-TNF groups. To sum up TNF-alpha has a role in skeletal muscle dysfunction in OVX rats and TNF-alpha antagonist administration recovered it. But this modulation was not sufficient for total structural recovery.

While cellular levels of arginine greatly exceed the apparent Km for endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production is limited by availability of arginine. Results from this work have provided a unique understanding of endothelial NO production, showing that arginine regeneration, that is the recycling of citrulline back to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL), defines the essential source of arginine for NO production. Using RNA interference analysis, selective reduction of AS expression was shown to directly correspond with a diminished capacity of endothelial cells to produce NO, despite saturating levels of arginine in the medium. In addition, the viability of AS siRNA-treated endothelial cells was compromised due to apoptotic cell death.AS expression was also investigated in response to two major vascular effectors. Tumor necrosis factor (TNF)-alpha; which is known to impair endothelial NO production, was shown to provoke a dose-dependent reduction of AS expression that corresponded to a decrease in NO production. Furthermore, TNF-alpha was shown to suppress AS expression through a NFkappaB mediated pathway, which involves three essential Sp1 elements in the proximal AS gene promoter. On the other hand, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, troglitazone and ciglitazone, which are known to elicit a vascular protective response against TNF-alpha effects, were shown to coordinately induce NO production and AS expression via a PPARgamma response element in the distal AS gene promoter. Importantly, these PPARgamma agonists were shown to restore AS expression and NO production following down-regulation by TNF-alpha, consistent with their vascular protective properties.

Le muscle squelettique fait preuve d'une remarquable plasticité en réponse aux changements physiologiques, comme l'activité physique, et aux situations pathologiques. Il subit notamment une atrophie sévère lors de la cachexie qui accompagne diverses pathologies chroniques comme le cancer, le SIDA, etc. L'atrophie musculaire est aussi une composante de la sarcopénie qui survient lors du vieillissement normal, et se caractérise par un déclin de la force et de la masse musculaire. L'atrophie musculaire, qui entraîne une augmentation de la mortalité et diminue l'efficacité des traitements, constitue donc un problème de santé majeur.La fonte musculaire se caractérise par une altération de l'équilibre entre synthèse et dégradation protéiques dans les fibres adultes. Des taux particulièrement élevés de cytokines circulantes, dont le TNFα, qui affectent l'homéostasie du muscle via différentes voies de signalisation, semblent être à l'origine de l'atrophie. Les mécanismes de la réponse atrophique musculaire à ces taux circulants élevés sont cependant mal définis. Le TNFα a des effets complexes. Il peut activer de multiples voies de signalisation, parmi lesquelles l'induction de la synthèse de sphingolipides, et plus particulièrement de céramides, par la voie de novo et par l'activation des sphingomyélinases. Au niveau musculaire, les céramides sont connus pour leurs effets sur la signalisation de l'insuline, sur l'apoptose et sur la différenciation myogénique. Par contre, leur implication dans le cadre de l'atrophie n'avait jamais été prise en compte. L'objectif de ce travail a été dans un premier temps de démontrer le rôle des céramides dans l'atrophie. Dans un deuxième temps, nous avons caractérisé la voie de signalisation par laquelle l'augmentation intramusculaire de céramide induite par le TNFα aboutit à une chute de la synthèse protéique, couplée à une augmentation de la protéolyse. Dans ce but, nous avons mis au point des modèles in vitro d'atrophie, impliquant des myotubes traités par des concentrations physiologiques de TNF. Nous avons en parallèle étudié un modèle in vivo de cachexie induite chez la souris par l'implantation d'un adénocarcinome C26. L'analyse des sphingolipides nous a permis de montrer l'augmentation des taux de céramides concomitante à l'atrophie générée in vitro et in vivo. Le rôle des céramides dans l'atrophie a été démontré par l'effet protecteur des inhibiteurs de leur synthèse, dans les modèles in vitro et in vivo. Nous montrons de plus dans un modèle in vitro que les effets atrophiques des céramides sont dus à l'inhibition de la voie de signalisation Phospholipase D/mTOR/Akt. Nos résultats nous ont permis de prouver le rôle des sphingolipides dans le contrôle de l'homéostasie protéique du muscle. La modulation du métabolisme des sphingolipides apparaît donc comme une nouvelle cible thérapeutique prometteuse dans le traitement de la perte musculaire associée à diverses pathologies.

The sphingolipids are a family of membrane lipids with only a structural role, influencing lipid bilayer properties, but they also act as effector molecules with essential roles in many aspects of cell biology. The sphingolipids ceramide, sphingosine and S1P have shown opposite effects: whereas ceramide and sphingosine usually inhibit proliferation and promote apoptotic responses to different stress stimuli, S1P is known to stimulate cell growth, and promote cell survival. Ceramide can be produced through the de novo synthesis pathway, and by membrane sphingomyelin hydrolysis catalyzed by sphingomyelinases. Both pathways can be activated by the pro-inflammatory cytokine TNFa. Because this cytokine has been shown to promote muscle loss and seems to be crucial in the development of cachexia, we hypothesized that the formation of ceramide, or a metabolite, can be involved in tumor-induced muscle wasting. We investigated the role of ceramide in the in vitro atrophic effects of TNFa on differentiated C2C12 myotubes, by using cell permeant ceramides and inhibitors of sphingolipid metabolism. We observed that TNFa atrophic effects, as evaluated by the reduction in myotube area, are mimicked by exogenous ceramides, supporting the idea that ceramide can participate in muscle atrophy. To verify if ceramide is a mediator of TNFa-induced atrophy, and to identify the metabolites potentially involved, we analyzed the effects of drugs able to block sphingolipid metabolism at different steps: the inhibition of de novo synthesis pathway was unable to restore myotube size in the presence of TNFa whereas the inhibitors of neutral sphingomyelinases reversed TNFa-induced atrophy. Moreover, an accumulation of ceramide and sphingosine induced pro-atrophic effects, whereas sphingosine-1-phosphate had a protective effect. These observations establish that in C2C12 myotubes, ceramide or other downstream metabolites such as sphingosine, produced by the neutral sphingomyelinase pathway in response to TNFa stimulation, participate in cell atrophy. To evaluate the in vivo role of sphingolipids, we treated BalbC mice carrying C26 adenocarcinoma woth Myriocin, an inhibitor of the de novo pathway of ceramide synthesis, that is able to deplete muscle tissue in all sphingolipids, was administered daily to the animals. This treatment partially protected animals against tumor-induced loss of body weight and muscle weight, without affecting the size of tumors. Moreover, myriocin treatment significantly reversed the decrease in myofiber size associated with tumor development, and reduced the expression of atrogenes Foxo3 and Atrogin-1, showing that it was able to protect against muscle atrophy. These results strongly suggest that ceramide, or a downstream sphingolipid metabolite, is involved in tumor-induced muscle atrophy. The sphingolipid pathway thus appears as a new potential target of pharmacological interventions aiming at protecting muscle tissue against atrophy.

The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the unidecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5 (Acetylamino) 4 oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4 phenyl 3 butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA Me and PBA both inhibit SP stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA Me and PBA dock at the ATP binding site of apoptosis signal regulating kinase 1 (ASK1) with predicted docking energies of -7.0 kcal/mol and 5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings support the conclusion that AOPHA Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule. We also encapsulated AOPHA-Me separately in polyketal and poly(lactic co glycolic acid) microparticles. The in-vitro release profiles of AOPHA-Me from these particles were characterized. We have also shown that AOPHA-Me, when encapsulated in PCADK microparticles, is an effective treatment for edema induced by adjuvant arthritis in rats. In separate work, it was determined that myo inositol 1,2,3,4,5,6 hexakisphosphate is an inhibitor to early-stage Loblolly pine somatic embryo growth. In addition, it was determined that muco inositol 1,2,3,4,5,6 hexakisphosphate is not an inhibitor to early-stage Loblolly pine somatic embryo growth. These experiments demonstrate the stereochemical dependence of myo inositol 1,2,3,4,5,6 hexakisphosphates inhibitory activity.

Tenocytes are the principal cells of the human Achilles tendon. In tendinosis, changes in the metabolism and morphology of these cells occur. Neurotrophins are growth factors essential for the development of the nervous system. Tumour necrosis factor alpha (TNF-α) has been found to kill sarcomas but has destructive effects in several major diseases. The two systems have interaction effects and are associated with apoptosis, proliferation, and pain signalling in various diseases. Whether these systems are present in the Achilles tendon and in Achilles tendinosis is unknown. The hypothesis is that the tenocytes produce substances belonging to these systems. In Studies I–III, we show that the potent effects of these substances are also likely to occur in the Achilles tendon. We found tenocyte immunoreactions for the neurotrophins brain-derived neurotrophic factor (BDNF), the nerve growth factor (NGF), the neurotrophin receptor p75, and for TNF-α and both of its receptors, TNFR1 and TNFR2. This occurred in both subjects with painful mid-portion Achilles tendinosis, and in controls. Furthermore, we found mRNA expression for BDNF and TNF-α in tenocytes, which proves that these cells produce these substances. TNFR1 mRNA was also detected for the tenocytes, and TNFR1 immunoreactions were upregulated in tendinosis tendons. This might explain why tenocytes in tendinosis undergo apoptosis more often than in normal tendons. Total physical activity (TPA) level and blood concentration of both soluble TNFR1 and BDNF were measured in Study IV. The results showed that the blood concentration of both factors were similar in subjects with tendinosis and in controls. Nevertheless, the TPA level was related to the blood concentration of sTNFR1 in tendinosis, but not in controls. This relationship should be studied further. The findings of this doctoral thesis show that neurotrophin and TNF-α systems are expressed in the Achilles tendon. We believe that the functions include tissue remodelling, proliferation and apoptosis.

Orientador: Ibsen Bellini Coimbra / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T21:15:58Z (GMT). No. of bitstreams: 1 Sartori_AngelicaRossi_D.pdf: 3838547 bytes, checksum: 0be815456ed0b3b8faf496ae6738da31 (MD5) Previous issue date: 2009 / Resumo: O Fator Induzido por Hipóxia-1 (Hypoxia Inducible Factor-1 - HIF-1) é um fator de transcrição responsável por transcrever genes relacionados às alterações nas concentrações de oxigênio e sobrevivência celular. A cartilagem articular é um tecido avascular e o ambiente dos condrócitos é caracterizado por condições de hipóxia dentro da matriz. Nestas condições a proteína HIF-1alfa do Fator de transcrição Induzido por Hipóxia-1alfa (HIF-1alfa) é necessário para controlar o metabolismo e a integridade funcional da cartilagem. Além da hipóxia algumas citocinas como IL-1? e TNF? são também capazes de estabilizar HIF-1?, além de serem consideradas as principais mediadoras da osteoartrite (OA). Objetivo: Verificar a participação da IL-1? na regulação do HIF-1? em condições normais de oxigênio; Verificar a utilização da via da Fosfatidil-Inositol-3-Kinase (PI-3K) pelo HIF-1? e analisar a participação HIF-1? na expressão de colágeno tipo II e agrecano e a sua regulação pelas citocinas TNF-? e da IL-1?. Material e Métodos: Condrócitos humanos provenientes de pacientes em OA, submetidos à artroplastia de joelho, foram cutivados em suspensão e em monocamada, submetidos ou não ao silenciamento do gene do HIF-1? pela técnica de Interferência por RNA e estimulados com IL-1?, TNF? e LY294002, o inibidor da via da PI-3K em condições normais de oxigênio e em hipóxia e foram submetidos à extração de proteína nuclear, extração de RNA e precipitação das proteínas do meio de cultura das células. A análise das proteínas foi feita por meio da técnica de Western Blotting para a detecção da proteína nuclear HIF-1? e do colágeno tipo II no meio de cultura das células. O RNA foi analisado pela técnica de PCR em Tempo Real para a quantificaçãos dos genes do HIF-1?, Colágeno tipo II e Agrecano. Resultados: IL-1? aumentou a expressão da proteína nuclear HIF-1?, mas não alterou a expressão do RNAm. Essa modulação utilizou a via da PI-3K. A hipóxia aumentou as concentrações do RNAm do HIF-1? em comparação com as condições normais de oxigênio, mas IL-1? e TNF? não alteraram o RNAm do HIF- 1? em condições normais de oxigênio e em associação com a hipóxia, inibiram o efeito regulatório positivo desta sobre o HIF-1?. A hipóxia isoladamente também aumentou RNAm de colágeno tipo II, o que foi anulado pelo estímulo associado com a IL-1?. Nos grupos de condrócitos com deficiência do HIF-1? (silenciados), em quaisquer condições de oxigênio, IL-1? e TNF? não alteraram significativamente a expressão de HIF-1?, mas em hipóxia, a expressão de colágeno tipo II foi regulada negativamente nesses grupos. A associação entre a falta do HIF-1? e as citocinas diminuiu ainda mais a expressão do colágeno tipo II em condições de hipóxia. Em todas as análises não foram observadas diferenças significativas nas expressões do RNAm do agrecano e na análise do colágeno tipo II das proteínas precipitadas dos meios de cultura das células. Conclusão: IL-1? aumentou a expressão da proteína nuclear HIF-1? pós-transcrição. A regulação do HIF-1? pela IL-1? em situação normal de oxigênio ocorreu, ao menos em parte, pela PI-3K. O HIF-1? se relacionou positivamente com a expressão do gene do colágeno tipo II, principalmente em hipóxia, mas não com os níveis da proteína. Não houve associação com a expressão do gene do agrecano / Abstract: Introduction: Hypoxia Inducible Factor -1 (HIF-1) is a transcription factor that regulates the expression of genes related with oxygen concentration e cellular survive. Articular cartilage is a non-vascular tissue and the condrocytes microenviroment are caracterized by hypoxic conditions inside the extracelluar matrix. In this condition, the protein HIF-1?, from HIF-1 is necessary to the metabolism control and cartilage functional intengrity. Some citokines like IL-1? and TNF?, as well as hypoxia, are capable to stabilize HIF-1? and are essential in oateoarthritis (OA) progression disease. Objective: To verify the participation of IL-1? in the HIF-1? regulation under normal conditions of oxygen; To verify if the pathway of phosphatidilynositol-3-Kinase (PI-3K) is used in this modulation; To analyse HIF-1? participation in the collagen type II and aggrecan gene expression and your regulation by TNF-? and IL-1?. Material and Methods: Human OA chondrocytes were obtained from patients with OA that underwent total knee joint replacement surgery, were cultured either in suspension or monolayer. They were submitted to HIF-1? gene silencing by RNA Interference and lately stimulated with IL-1?, TNF? and LY294002, a specific inhibitor to Phosphatidilinosiltol - 3- Kinase (PI-3K) pathway in normal conditions of oxygen and in hypoxia. The condrocytes were submitted to protein nuclear extraction, RNA extraction and protein precipitation of culture media from the experiments. The proteins were analyzed by Western Blotting to HIF-1? detection in nuclear extraction and collagen type II detection in precipitated media. The RNA were analized by Real Time PCR to HIF- 1?, collagen type II and aggrecan gene quantification. Results: HIF-1? expression is up-regulated by IL-1? at the protein level but not in the mRNA expression. This modulation used, at least in part, the PI-3K pathway. Hypoxia enhanced the mRNA concentrations of HIF-1? when compared with normal conditions of oxygen, but in this cases IL-1? and TNF? did not change mRNA expression of HIF-1?. In association with hypoxia, these citokines inhibited the positive regulatory effect under HIF-1? mRNA expression. Hypoxia up-regulated collagen type II gene expression and that was inhibited by the association with IL-1?. In the groups with lack of HIF-1? (silenced), in both oxygen conditions, IL-1? and TNF? did not cause any significant change of the HIF-1? mRNA expression, but in hypoxia, collagen type II was up-regulated in those groups. The association between the lack of HIF- 1? and citokines down-regulated more strongly collagen type II in hypoxia. In all analyzes it was not observed significant differences in the aggrecan mRNA expression and collagen tipe II protein from cultured media of the cells Conclusion: IL-1? post-trancriptionaly up-regulated the HIF-1? protein level. HIF- 1? regulation by IL-1? in normal conditions of oxygen used, at least in part, the PI- 3K pathway. HIF-1? positively related with gene expression, but not with the protein levels, of collagen type II, mainly in hypoxia. There was no relation in the HIF-1? and aggrecan gene expression / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Master of Public Health / Department of Diagnostic Medicine/Pathobiology / John A. Pickrell / Beef cattle feed lots produce significant, local point source pollution of the atmosphere. The dusts generated in the CAFOs are complex mixture of fine and ultra fine particles, organic compounds, transition metals, and adsorbed toxic gases. Since each component is toxic in itself, we do not fully understand the relative importance of each component in the dust and their interactions to inducing inflammatory changes in the lung. We did extensive literature searches to understand the mechanism of dust toxicity in respiratory system. This lead to focusing on solubility of dust in lung simulant fluid, and in-vitro study of release of two common biomarkers of inflammatory processes IL-6 and IL-8 from lung epithelial cells. Various concentrations (1 to 50%) of the dust extract induced release of IL-6, and IL-8 from lung epithelial cell as indicators of pro-inflammatory changes (IL-6), and amplification and maintenance of inflammation (IL-8). IL-6 release had dose dependence; peak production was seen with 25% dust extract. IL-8 production went down as the concentration of the dust extract increased from 1% to 25%. However, 50% dust extract was cytotoxic to the cell leading to 10-15% cell viability. At non-cytotoxic concentrations for lung epithelial cells, production of IL-8 was reduced. These findings suggested that higher exposure concentration were required to initiate inflammation as indicated by IL-6 release. Lower exposure concentrations (1 and 5% extracts) were related to optimal release of IL-8 needed to amplify and maintain the inflammatory response. Inhibition of endotoxin didn't significantly change the pattern of IL-6 or IL-8 release from epithelial cells. This finding suggested that at least a portion of the mechanism by which particle induced cytokine release from the lung epithelial cells was not endotoxin dependent. Heating samples at 1200C for 5 minutes modified some of the toxic properties of the dust extracts but didn't completely detoxify it. We observed that longer incubation period was required to peak release for both IL-6 and IL-8. However, the higher concentration of sample (50% extract) found to be cytotoxic in non-heat treated sample was no longer cytotoxic and induced both IL-6 and IL-8 release from the lung epithelial cells. This result suggested that heat treatment could reduce some of the dust extract's cytotoxic properties. However, the extract's potential to induce peak cytokine release increased.

Painful conditions having the origin within the musculoskeletal system is a common cause for people to seek medical care. Between 20-40% of all visits to the primal care in Sweden are coupled to pain from the musculoskeletal system. Muscle pain and impaired muscle function can be caused by muscles being repetitively overused and/or via heavy load. Skeletal muscle is a dynamic tissue which can undergo changes in order to fulfill what is best for optimal function. However, if the load is too heavy, morphological changes including necrosis, as well as pain can occur. The extension of the skeletal muscle is the tendon. Tendinopathy refers to illness and pain of the tendon. The peritendinous tissue is of importance in the features related to tendon pain. Common tendons/origins being afflicted by tendinopathy/pain are the Achilles tendon and the extensor origin at the elbow region.    Tumor necrosis factor alpha (TNF-alpha) is a cytokine that is involved in several biological processes. It is well-known for its involvement in the immune system and is an important target for inflammatory disorders such as rheumatoid arthritis. It is not known to what extent the TNF-alpha system is involved in the process of muscle inflammation and damage due to overuse.    Studies were conducted on rabbit and human tissue, tissues that either had undergone an excessive loading activity or tissue that was removed with surgery due to painful conditions. The tissues were evaluated via staining for morphology, in situ hybridization and immunofluorescence.    Unilateral experimental overuse of rabbit muscle (soleus muscle) led to morphological changes in the soleus muscle tissue bilaterally. The longer the experiment extended, the more was the tissue affected. This included infiltration of white blood cells in the tissue (myositis) and abnormal muscle fiber appearances. TNF-alpha mRNA was seen in white blood cells, in muscle fibers interpreted to be in a reparative stage and in white blood cells that had infiltrated into necrotic muscle fibers.  There was an upregulation in expressions of TNF receptor type 1 (TNFR1) and TNF receptor type 2 (TNFR2) in muscles that were markedly overused, with expressions in white blood cells, fibroblasts, blood vessel walls and muscle fibers. Immunoreactions for the receptors were seen in nerve fascicles of markedly overused muscles but only occasionally in normal muscles. The upregulations were seen for both experimental and contralateral sides. Overall the two receptors showed somewhat different expression patterns. Tendinopathy is associated with an increase in blood flow and infiltration of white blood cells in the tissue adjacent to the tendon. It is called the peritendinous tissue and is also richly innervated. The white blood cells and the blood vessels walls in this tissue were showing immunoreaction for TNFR1 and TNFR2. Two types of nerve fascicles were found in this tissue, one normally appearing when staining for nerve markers and one type with signs of axonal loss. The latter had clearly strong immunoreactions for TNFR1 and TNFR2.    The findings suggest that the TNF-alpha system is involved in both myopathies occurring due to overuse and in features in the peritendinous tissue in the tendinopathy situation. TNF-alpha and its receptors seem to be involved in degeneration but also in regeneration and healing of the tissue. The findings also suggest that TNF-alpha has effects on nerves showing axonal loss. The changes in the TNF-alpha system were seen both on the experimental side and contralaterally. / Smärta och funktionsbortfall från rörelseapparaten är vanligt förekommande. Mellan 20-40% av alla besök i primärvården är kopplade till smärta från rörelseapparaten. Det är också en vanlig orsak till sjukfrånvaro. Överansträngning inklusive repetitivt enformigt muskelarbete kan leda till muskelsmärta och bristande muskelfunktion (ex nedsatt styrka och uthållighet, inskränkt rörlighet). Muskelvävnad är en dynamisk vävnad som kan ändras utefter vilka påfrestningar den utsätts för och därigenom vilka behov den ställs inför. Men om belastningen blir för hård, alternativt återhämtningen blir för kort, kan negativa förändringar i vävnadsstrukturen uppstå, inklusive celldöd och vävnadsskada. Förlängningen av muskeln är senan. Senan är den vävnad som förbinder muskeln med skelettet. Tendinopati innefattar smärtsamma sjukdomstillstånd i senan. När sjukdom i en sena uppstår, exempelvis en smärtande hälsena, har man sett att den lösa bindväven som omger senan är av betydelse. Den genomgår morfologiska förändringar och man tror att det är den som är med och bidrar till smärtan vid tillståndet. Akillessenan och ”tennis-armbåge” är vanliga ställen för tendinopati. Akillessenan förbinder den trehövdade vadmuskeln med hälbenet. Tennis-armbåge omfattar ett område för flera musklers ursprung vid armbågen. Dessa muskler ansvarar framför allt för att sträcka i handleden. TNF-alfa är en signalsubstans som är involverad i flertalet biologiska processer. Den är känd för sin del i immunförsvaret och den är ett viktigt mål för behandling av autoimmuna sjukdomar som exempelvis reumatoid artrit. Det är inte känt om TNF-alfa är inblandad i processen som uppstår vid muskelinflammation/muskelskada efter kraftig överansträngning. TNF-alfa har flera receptorer, i det här arbetet har utbredning av TNFR1 och TNFR2 analyserats. Studier har utförts på djur (kaniner) och människa. Kaniner har genomgått ett träningsexperiment, där de utsatts för repetitiva muskelkontraktioner som lett till överansträngningsskador och muskelinflammation. Den muskel som studerats är soleus-muskeln, en del i den trehövdade vadmuskeln. Vävnadsprover har tagits från patienter med smärta i Akillessenan eller tennisarmbåge. Vävnadsproverna från kanin och människa har analyserats med färgningar för morfologi, immunohistokemi för detektering av TNF-alfa och dess receptorer samt för in situ hybridisering för detektion av mRNA i TNF-alfa systemet. Parallellt med färgningar för faktorerna i TNF-alfa systemet har uttryck för andra faktorer studerats. Ensidig överbelastning hos kaniner ledde till samma morfologiska förändringar på båda sidor, det vill säga även i muskeln i det ben som inte hade genomgått träningsexperimentet. Ju längre experimentet pågick, desto större blev de morfologiska förändringarna. TNF-alfa sågs i vita blodkroppar, TNF-alfa mRNA sågs även i förändrade muskelfibrer. Resultatet av parallella dubbelfärgningar tolkades som att dessa muskelfibrer antingen var i en regenererande process eller i en destruktiv process. TNFR1 och TNFR2 uttrycktes i större utsträckning ju längre experimentet pågick och ju mer muskelvävnaden var påverkad av inflammation. TNF receptorer sågs i vita blodkroppar, fibroblaster, muskelfibrer och nervstrukturer hos experimentdjuren. Det såg lika ut på båda sidor, inklusive det ben som inte ingått i experimentet. De två receptorerna skilde sig åt i uttryck. Vävnad från patienter med smärtande senor/smärta vid muskelursprungs-region genomgick också färgningar för faktorer i TNF-alfa systemet. Man kunde se att den lösa bindväven runt senan (den peritendinösa vävnaden) innehöll mycket blodkärl och nerver. De nerver som sågs i denna vävnad var av två typer, en som såg normal ut och en typ som uppvisade tecken på förlust av axoner. Den senare varianten hade en tydlig uppreglering av båda TNF receptorerna. Dessa resultat tyder på att TNF-alfa systemet är involverat i muskelsjukdomar som rör muskelinflammation till följd av kraftig överansträngning och i processerna i bindväven vid smärtande senor. TNF-alfa och dess receptorer verkar vara inblandade i både nedbrytning och uppbyggnad av muskelvävnad, samt påverka nerver som visar tecken på förlust av axoner. Förändringarna i TNF-alfa systemet sågs både på experimentsidan och kontralateralt.