Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2006. / Härtill 4 uppsatser.

Pain of the musculoskeletal system is one of the most common reasons for people seeking medical attention, and is also one of the major factors that prevent patients from working. Chronic tendon pain, tendinopathy, affects millions of workers world-wide, and the Achilles tendon is an important structure often afflicted by this condition. The pathogenesis of tendinopathy is poorly understood, but it is thought to be of multifactoral aetiology. It is known that tendon pain is often accompanied not only by impaired function but also by structural tissue changes, like vascular proliferation, irregular collagen organisation, and hypercellularity, whereby the condition is called tendinosis. In light of the poor knowledge of tendinosis pathophysiology and recent findings of a non-neuronal signalling system in tendon tissue, the contributory role of neuropeptides such as substance P (SP) has gained increased interest. SP, known for afferent pain signalling in the nervous system, also has multiple efferent functions and has been described to be expressed by non-neuronal cells. As pain is the most prominent symptom of tendinopathy, the focus of the studies in this thesis was the innervation patterns of the tissue ventral to the Achilles tendon (i.e. the tissue targeted in many contemporary treatment methods) as well as the distribution of SP and its preferred receptor, the neurokinin-1 receptor (NK-1R), in the tendon tissue itself. It was hereby hypothesised that the source of SP affecting the Achilles tendon might be the main cells of the tendon tissue (the tenocytes) as well as paratendinous nerves, and that SP might be involved in tendinosis- development. The studies were conducted, via morphological staining methods including immunohistochemistry and in situ hybridisation, on tendon biopsies from patients suffering from Achilles tendinosis and on those from healthy volunteers. The hypothesis of the thesis was furthermore tested using an experimental animal model (rabbit) of Achilles tendinopathy, which was first validated. The model was based on a previously established overuse protocol of repetitive exercise. In the human biopsies of the tissue ventral to the Achilles tendon, there was a marked occurrence of sympathetic innervation, but also sensory, SP-containing, nerve fibres. NK-1R was expressed on blood vessels and nerve fascicles of the paratendinous tissue, but also on the tenocytes of the tendon tissue proper itself, and notably more so in patients suffering from tendinosis. Furthermore, the human tenocytes displayed not only NK-1R mRNA but also mRNA for SP. The animal model was shown to produce objectively verified tendinosis-like changes, such as hypercellularity and increased vascularity, in the rabbit Achilles tendons, after a minimum of three weeks of the exercise protocol. The contralateral leg of the animals in the model was found to be an unreliable control, as bilateral changes occured. The model furthermore demonstrated that exogenously administered SP triggers an inflammatory response in the paratendinous tissue and accelerates the intratendinous tendinosis-like changes such that they now occur after only one week of the protocol. Injections of saline as a control showed similar results as SP concerning hypercellularity, but did not lead to vascular changes or pronounced paratendinous inflammation. In summary, this thesis concludes that interactions between the peripheral sympathetic and sensory nervous systems may occur in Achilles tendinosis at the level of the ventral paratendinous tissue, a region thought to be of great importance in chronic tendon pain since many successful treatments are directed toward it. Furthermore, the distribution of NK-1R:s in the Achilles tendon described in these studies gives a basis for SP, whether produced by nerves mainly outside the tendon or by tenocytes within the tendon, to affect blood vessels, nerve structures, and/or tendon cells, especially in tendinosis patients. In light of this and of previously known SP-effects, such as stimulation of angiogenesis, pain signalling, and cell proliferation, the proposed involvement of SP in tendinosis development seems likely. Indeed, the animal model of Achilles tendon overuse confirms that SP does induce vascular proliferation and hypercellularity in tendon tissue, thus strengthening theories of SP playing a role in tendinosis pathology.

Thesis (Ph.D.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2009/06/05). Includes bibliographical references.

Intravenous injection of substance P (SP) increases renal nerve firing and heart rate in spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) by stimulating sympathetic ganglia. Blood pressure is increased in SHRs but lowered in WKYs. This study assesses the role of neurokinin-1 (NK1) receptors in mediating the ganglion actions of SP. Rats for functional studies were anesthetized and then treated with chlorisondamine. Renal nerve, blood pressure, and heart rate responses to intravenous injection of the NK1 receptor agonist GR-73632 were similar but less than those to equimolar doses of SP in SHRs. GR-73632 only slightly increased renal nerve firing and heart rate and lowered blood pressure in WKYs. The NK1 receptor antagonist GR-82334 (200 nmol/kg iv) blocked the ganglionic actions of GR-73632 and the pressor response to SP in SHRs. It reduced the renal nerve and heart rate responses by 52 and 35%. This suggests that the pressor response to SP is mediated by ganglionic NK1 receptors and that NK1 receptors also have a prominent role in mediating the renal nerve and heart rate responses to SP. Quantitative autoradiography showed that NK1 receptors are more abundant in the superior cervical ganglia of SHRs. RT-PCR showed increased abundance of NK1 receptor mRNA in SHRs as well. These observations suggest that the greater ganglionic stimulation caused by SP in SHRs is due to upregulation of NK1 receptors.

A substância P (SP) é um neuropeptídeo da família das taquicininas que regula numerosas funções biológicas por meio da ligação ao seu receptor altamente específico neuroquinina-1 (NK-1R). Este complexo SP/NK-1R está envolvido em diversos processos relacionados à oncogênese, como a mitogênese, angiogênese, migração celular e metástase. O objetivo deste trabalho foi investigar a expressão de substância P e de seu receptor NK-1 e sua correlação com o índice de proliferação celular em 73 pacientes portadores de 90 carcinomas espinocelulares de boca, diagnosticados e tratados no Hospital General e Hospital de La Princesa, Jaen, Espanha, durante o período de 1995 a 2008. Todos os tumores foram corados pela técnica imunoistoquímica da estreptavidina-biotina-peroxidase com os anticorpos anti-SP, anti-NK-1R e anti-Ki-67. As alterações celulares epiteliais das margens cirúrgicas livres de doença também foram registradas. A expressão imunoistoquímica da substância P e do seu receptor neurokinina-1 foi avaliada na membrana, no citoplasma e no núcleo das células epiteliais malignas e do epitélio da mucosa bucal adjacente ao tumor, nos linfócitos e nos vasos sanguíneos dos tumores. O índice de proliferação celular tumoral foi determinado pela expressão imunoistoquímica de Ki-67 identificada no núcleo das células neoplásicas. As correlações entre as diversas localizações da SP, de seu receptor NK-1R e do índice de proliferação tumoral determinado pelo Ki-67 foram determinadas estatísticamente utilizando-se o Crosstab, Regress e Descript de SUDAAN. A expressão de SP foi identificada no estroma de 77% dos tumores, na membrana de 71% das células malignas e no citoplasma de 81,2% dos tumores. A maioria dos tumores apresentou altas taxas de proliferação das células neoplásicas com mais de 50% das células imunopositivas para o Ki-67. Ao analisar as margens cirúrgicas livres de doença, observou-se expressão da SP, sobretudo no terço inferior e médio, tanto no núcleo, como no citoplasma e na membrana celular. A expressão concomitante de substância P e do receptor neurokinina-1 no citoplasma das células neoplásicas ocorreu mais frequentemente nas células tumorais em proliferação. Verificou-se que expressão de SP no câncer bucal ocorre juntamente com o aumento da expressão de NK-1R, sugerindo que as células neoplásicas epiteliais bucais podem utilizar esta via para tornarem-se mais susceptíveis aos estímulos mediados pela SP. A expressão de substância P nos linfócitos do infiltrado inflamatório e vasos sanguíneos intratumorais e peritumorais se associaram a tumores de menor tamanho, menor estádio clínico e com menor frequência metástase ganglionar. Concluiu-se que as células neoplásicas epiteliais bucais podem utilizar a via substância P/NK-1R para tornarem-se mais susceptíveis aos estímulos mediados pela SP, particularmente aqueles associados à proliferação celular. Além disso, a expressão epitelial, citoplasmática e nuclear da substância P é um evento precoce na carcinogênese bucal podendo ser considerado um marcador da presença e intensidade de displasia epitelial. / The substance P (SP) is a neuropeptide of the tachykinin family that regulates multiple biological functions by binding to the highly specific receptor neurokinin-1. This complex SP/NK-1 is involved in several processes related to oncogenesis, such as mitogenesis, angiogenesis, cell migration and metastasis. This study investigated the expression of substance P and its receptor NK-1 and its correlation with the cell proliferation index in 73 patients with oral squamous cell carcinoma, diagnosed and treated at the General Hospital and Princess Hospital at Jaen, Spain, during the period 1995 to 2008. All tumors were stained immunohistochemically by the streptavidin-biotin-peroxidase technique using the antibodies anti-SP, anti-NK-1R and anti-Ki-67. The epithelial cell alterations on the disease-free surgical margins were registered. The immunohistochemical expression of SP and its receptor neurokinin-1 were evaluated on the membrane, cytoplasm and nucleus of malignant epithelial cells and cells of healthy oral mucosa adjacent to the tumor, as well as on the infiltrating lymphocytes and peritumoral or intratumoral blood vessels. The tumor cell proliferation index was determined by the immunohistochemical expression of Ki- 67 identified on the malignant cell nucleus. The correlations between the distinct localizations of SP, its receptor NK-1 and the proliferation index Ki-67 were statistically analyzed using the Sudaan Crosstab, Regress and Descript tests. The SP expression was identified on the stroma of 77% of tumors, on the membrane of 71% of malignant cells and cytoplasm of 81.2% of tumors. Most tumors presented high proliferation rates of neoplastic cells, with more than 50% of cells immunopositive for Ki-67. Analysis of the disease-free surgical margins revealed SP expression especially on the lower and medium third, both on the nucleus, cytoplasm and cell membrane. The simultaneous expression of substance P and its receptor NK-1 on the cytoplasm of neoplastic cells occurred more frequently in proliferating malignant cells. The expression of SP in oral cancer occurred simultaneously to an increased expression of NK-1R, suggesting that the oral malignant epithelial cells might use this pathway to become more susceptible to the stimuli mediated by the SP. The substance P expression on infiltrating lymphocytes and intratumoral or peritumoral blood vessels was associated with tumors of small size, lower clinical stage and less frequent node metastasis. It was concluded that the oral neoplastic epithelial cells may use the pathway SP/NK-1R to become more susceptible to the stimuli mediated by the SP, particularly those associated with cell proliferation. Additionally, the epithelial, cytoplasmic and nuclear expression of substance P is an early event in oral carcinogenesis and may be considered a marker of the presence and intensity of epithelial dysplasia.

A substância P (SP) é um neuropeptídeo da família das taquicininas que regula numerosas funções biológicas por meio da ligação ao seu receptor altamente específico neuroquinina-1 (NK-1R). Este complexo SP/NK-1R está envolvido em diversos processos relacionados à oncogênese, como a mitogênese, angiogênese, migração celular e metástase. O objetivo deste trabalho foi investigar a expressão de substância P e de seu receptor NK-1 e sua correlação com o índice de proliferação celular em 73 pacientes portadores de 90 carcinomas espinocelulares de boca, diagnosticados e tratados no Hospital General e Hospital de La Princesa, Jaen, Espanha, durante o período de 1995 a 2008. Todos os tumores foram corados pela técnica imunoistoquímica da estreptavidina-biotina-peroxidase com os anticorpos anti-SP, anti-NK-1R e anti-Ki-67. As alterações celulares epiteliais das margens cirúrgicas livres de doença também foram registradas. A expressão imunoistoquímica da substância P e do seu receptor neurokinina-1 foi avaliada na membrana, no citoplasma e no núcleo das células epiteliais malignas e do epitélio da mucosa bucal adjacente ao tumor, nos linfócitos e nos vasos sanguíneos dos tumores. O índice de proliferação celular tumoral foi determinado pela expressão imunoistoquímica de Ki-67 identificada no núcleo das células neoplásicas. As correlações entre as diversas localizações da SP, de seu receptor NK-1R e do índice de proliferação tumoral determinado pelo Ki-67 foram determinadas estatísticamente utilizando-se o Crosstab, Regress e Descript de SUDAAN. A expressão de SP foi identificada no estroma de 77% dos tumores, na membrana de 71% das células malignas e no citoplasma de 81,2% dos tumores. A maioria dos tumores apresentou altas taxas de proliferação das células neoplásicas com mais de 50% das células imunopositivas para o Ki-67. Ao analisar as margens cirúrgicas livres de doença, observou-se expressão da SP, sobretudo no terço inferior e médio, tanto no núcleo, como no citoplasma e na membrana celular. A expressão concomitante de substância P e do receptor neurokinina-1 no citoplasma das células neoplásicas ocorreu mais frequentemente nas células tumorais em proliferação. Verificou-se que expressão de SP no câncer bucal ocorre juntamente com o aumento da expressão de NK-1R, sugerindo que as células neoplásicas epiteliais bucais podem utilizar esta via para tornarem-se mais susceptíveis aos estímulos mediados pela SP. A expressão de substância P nos linfócitos do infiltrado inflamatório e vasos sanguíneos intratumorais e peritumorais se associaram a tumores de menor tamanho, menor estádio clínico e com menor frequência metástase ganglionar. Concluiu-se que as células neoplásicas epiteliais bucais podem utilizar a via substância P/NK-1R para tornarem-se mais susceptíveis aos estímulos mediados pela SP, particularmente aqueles associados à proliferação celular. Além disso, a expressão epitelial, citoplasmática e nuclear da substância P é um evento precoce na carcinogênese bucal podendo ser considerado um marcador da presença e intensidade de displasia epitelial. / The substance P (SP) is a neuropeptide of the tachykinin family that regulates multiple biological functions by binding to the highly specific receptor neurokinin-1. This complex SP/NK-1 is involved in several processes related to oncogenesis, such as mitogenesis, angiogenesis, cell migration and metastasis. This study investigated the expression of substance P and its receptor NK-1 and its correlation with the cell proliferation index in 73 patients with oral squamous cell carcinoma, diagnosed and treated at the General Hospital and Princess Hospital at Jaen, Spain, during the period 1995 to 2008. All tumors were stained immunohistochemically by the streptavidin-biotin-peroxidase technique using the antibodies anti-SP, anti-NK-1R and anti-Ki-67. The epithelial cell alterations on the disease-free surgical margins were registered. The immunohistochemical expression of SP and its receptor neurokinin-1 were evaluated on the membrane, cytoplasm and nucleus of malignant epithelial cells and cells of healthy oral mucosa adjacent to the tumor, as well as on the infiltrating lymphocytes and peritumoral or intratumoral blood vessels. The tumor cell proliferation index was determined by the immunohistochemical expression of Ki- 67 identified on the malignant cell nucleus. The correlations between the distinct localizations of SP, its receptor NK-1 and the proliferation index Ki-67 were statistically analyzed using the Sudaan Crosstab, Regress and Descript tests. The SP expression was identified on the stroma of 77% of tumors, on the membrane of 71% of malignant cells and cytoplasm of 81.2% of tumors. Most tumors presented high proliferation rates of neoplastic cells, with more than 50% of cells immunopositive for Ki-67. Analysis of the disease-free surgical margins revealed SP expression especially on the lower and medium third, both on the nucleus, cytoplasm and cell membrane. The simultaneous expression of substance P and its receptor NK-1 on the cytoplasm of neoplastic cells occurred more frequently in proliferating malignant cells. The expression of SP in oral cancer occurred simultaneously to an increased expression of NK-1R, suggesting that the oral malignant epithelial cells might use this pathway to become more susceptible to the stimuli mediated by the SP. The substance P expression on infiltrating lymphocytes and intratumoral or peritumoral blood vessels was associated with tumors of small size, lower clinical stage and less frequent node metastasis. It was concluded that the oral neoplastic epithelial cells may use the pathway SP/NK-1R to become more susceptible to the stimuli mediated by the SP, particularly those associated with cell proliferation. Additionally, the epithelial, cytoplasmic and nuclear expression of substance P is an early event in oral carcinogenesis and may be considered a marker of the presence and intensity of epithelial dysplasia.

This thesis is on chronic neck pain after a rear impact car injury, so called whiplash associated disorder (WAD). Three empirical studies using positron emission tomography (PET) with different radioligands have been performed. The first study evaluated resting state regional cerebral blood flow (rCBF) in WAD patients and in healthy, pain-free controls, by use of oxygen-15 labeled water. Patients had heightened resting rCBF bilaterally in the posterior parahippocampal and the posterior cingulate gyri, in the right thalamus and in the right medial prefrontal gyrus. Attenuated tempero-occipital blood flow was also observed in the patient group as compared to healthy controls. Alterations in rCBF were related to patients’ neck disability ratings. Study I suggests an involvement of the posterior cingulate, the parahippocampal and the medial prefrontal gyri in WAD. This altered resting state neural activity may be linked to an increased self-relevant evaluation of pain and stress. The second study evaluated central expression of the neurokinin-1 (NK1) receptor in WAD patients and healthy controls. Using a carbon-11 labeled specific NK1 antagonist, the receptor availability was measured. Patients displayed lowered NK1 receptor availability in the insula, anterior cingulate, frontal lobe, hippocampus, amygdala and in the periaqueductal gray matter, consistent with results from animal models of chronic pain. NK1 receptor availability was most reduced in the ventromedial orbitofrontal cortex, where attenuations were linearly related to patients fear and avoidance of movement. Thirdly, carbon-11 labeled D-deprenyl was used to investigate the presence of locally inflamed soft tissue in the cervical neck in WAD patients. Although the retention mechanism of [11C]D-deprenyl is not known, the results suggest that WAD patients have chronic inflammatory processes in the neck, most commonly in the adipose tissue at the spineous process of the second vertebra. In summary, this thesis provides evidence for altered central blood flow and receptor characteristics in WAD patients. Further, WAD patients may also have signs of persistent peripheral tissue damage. Both central and peripheral pain mechanisms have been demonstrated and visualized in patients with whiplash associated disorder.

abstract: Systemic lupus erytematosus (SLE) is an autoimmune disease where the immune system is reactive to self antigens resulting in manifestations like glomerulonephritis and arthritis. The immune system also affects the central nervous system (known as CNS-SLE) leading to neuropsychiatric manifestations such as depression, cognitive impairment, psychosis and seizures. A subset of pathogenic brain-reactive autoantibodies (BRAA) is hypothesized to bind to integral membrane brain proteins, affecting their function, leading to CNS-SLE. I have tested this BRAA hypothesis, using our lupus-mouse model the MRL/lpr mice, and have found it to be a reasonable explanation for some of the manifestations of CNS-SLE. Even when the MRL/lpr had a reduced autoimmune phenotype, their low BRAA sera levels correlated with CNS involvement. The correlation existed between BRAA levels to integral membrane protein and depressive-like behavior. These results were the first to show a correlation between behavioral changes and BRAA levels from brain membrane antigen as oppose to cultured neuronal cells. More accurate means of predicting and diagnosing lupus and CNS-SLE is necessary. Using microarray technology I was able to determine peptide sets that could be predictive and diagnostic of lupus and each specific CNS manifestation. To knowledge no test currently exists that can effectively diagnose lupus and distinguish between each CNS manifestations. Using the peptide sets, I was able to determine possible natural protein biomarkers for each set as well as for five monoclonal BRAA from one MRL/lpr. These biomarkers can provide specific targets for therapy depending on the manifestation. It was necessary to investigate how these BRAA enter the brain. I hypothesized that substance P plays a role in altering the blood-brain barrier (BBB) allowing these BRAA to enter and affect brain function, when bound to its neurokinin-1 receptor (NK-1R). Western blotting results revealed an increase in the levels of NK-1R in the brain of the MRL/lpr compared to the MRL/mp. These MRL/lpr with increased levels of both NK-1R and BRAA displayed CNS dysfunction. Together, these results demonstrate that NK-1R may play a role in CNS manifestations. Overall, the research conducted here, add to the role that BRAA are playing in CNS-lupus. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

Le récepteur neurokinine 1 (NK1R) est impliqué dans la régulation des réponses immunitaires innées et adaptatives. Cependant, les mécanismes par lesquels le NK1R modulerait ces réponses ne sont pas connus. Chez les cellules T, les voies de la calcineurine et de la mTOR constituent les cibles d’immunosuppresseurs, comme la cyclosporine A (CsA), le tacrolimus et la rapamycine. Ainsi, nous avons voulu déterminer si le NK1R pourrait agir sur ces voies et si le blocage pharmacologique du NK1R avec des antagonistes sélectifs, pourrait augmenter l’action de ces immunosuppresseurs sur l’activation des cellules T. Tout d’abord, nos résultats ont montré que les cellules Jurkat (celules T humaines) exprimaient à la fois le gène du NK1R et de son ligand (les endokinines). Ceci suggère l'existence d'une régulation autocrine tachykinergique de la fonction des cellules T. Cette hypothèse est appuyée par nos données, où nous avons observé que le blocage du NK1R avec des antagonistes spécifiques (L-733,060 et L-703,606) chez les cellules Jurkat, inhibe la production d'IL-2 et diminue l'activation du NFAT (substrat de la calcineurine). De façon intéressante, nous avons montré un effet de combinaison entre les antagonistes du NK1R et les inhibiteurs de la calcineurine (CsA et tacrolimus) sur la production d’IL-2 et l’activation du NFAT. En revanche, le blocage du NK1R n'a pas d'effet inhibiteur sur l’activation de la mTOR et la p70S6K, mais réduit la phosphorylation de S6R (Ser235/236) et Akt (Ser473). Enfin, nous n’avons observé aucun effet de combinaison avec la rapamycine et l’antagoniste NK1R sur l’activation de mTOR et de sa voie de signalisation. L’ensemble de nos résultats, démontrent la présence d'un nouveau mécanisme de régulation de NFAT impliquant le système tachykinergique NK1R/endokinines chez les cellules T. Par conséquent, nous suggérons que la combinaison des antagonistes NK1R avec les inhibiteurs de la calcineurine pourrait être une alternative thérapeutique intéressante afin de réduire les doses de CsA et le FK506 dans les protocoles de prévention de rejet de greffes. / There are increasing evidences for a role of the neurokinin 1 receptor (NK1R) in the regulation of innate and adaptive immune systems. However, whether NK1R regulates calcineurin/nuclear factor of activated T cell (NFAT) and mTOR pathways in T cells is unknown. These signalling pathways being targets of the immunosuppressive drugs cyclosporine A (CsA), FK506 and rapamycin respectively. We also examined whether pharmacological blockade of NK1R may be combined to those immunosuppressors to repress T cell activation. In this article, we first show that Jurkat T cells express both genes for NK1R and its ligands endokinins which suggests the existence of an autocrine tachykinergic regulation of T cells function. This hypothesis is supported by our data showing that blockade of this receptor with specific NK1R antagonists inhibits IL-2 production in Jurkat T cells which is associated with the reduction of NFAT activation. Interestingly, we show interplay between NK1R antagonists and calcineurin inhibitors to repress IL-2 production and NFAT activation. In contrast, blockade of NK1R has no inhibitory effect on mTOR and p70S6K activation but reduce S6R (Ser235/236) and Akt (Ser473) phosphorylation. However, combining rapamycin with NK1R antagonist has no enhancing effect on rapamycin-reduced mTOR activation and its signalling pathway. Our findings provide the evidence of a novel mechanism of regulation of NFAT activation-induced IL-2 production in T cells involving the tachykinergic system NK1R/endokinins. These observations may offer new application for NK1R antagonists in transplantation immunotherapy in combination with immunosuppressors.

No description available.