Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Abstract The Achilles tendon is the largest tendon in the human body and is affected by many diseases and is vulnerable to many forms of damage due to the heavy loads it must bear. Rupture of the Achilles tendon has become more common in recent times, with an almost four-fold increase in prevalence from 1979–1990 to 1991–2000 and a peak incidence of 19 ruptures per 100 000 of population in 1999 in our epidemiological assessment. The incidences of major complications, re-rupture and deep infection, increased along with primary ruptures, peaking in 1999. The results after successful primary repair are good in over 90% of cases, as we have shown in a randomized study and in a review of the literature, and the result after re-rupture is still good in about 70% of cases, but achieving good performance after deep infection is a highly random matter. Our retrospective survey did not identify any good results, but the deep infection cases in our randomized study showed good performance due to prompt action taken for their treatment. The best method for treating a ruptured Achilles tendon has been under debate for almost 100 years, with surgery and conservative methods advocated to equal extents. We have advocated surgical treatment as the primary choice and conservative treatment is given for selected high risk patients, for example patients with diabetes, skin problems, systemic use of corticosteroids or severe other illness. The type of surgery technique is not a straightforward choice, either, and various forms of open surgery and percutaneous techniques exist. We compared an end-to-end simple suture with the same suture augmented with one central gastrocnemius turn-over flap in a randomized series of 60 patients and found no differences with respect to subjective complaints, calf muscle strength or tendon elongation with time. The end-to-end technique is simpler and is therefore justified as the primary method of choice for the surgical repair of fresh complete Achilles tendon ruptures. The tissue composition has been shown to alter not only with time but also after repeated tearing of the tendon collagen fibres. A normal tendon is mainly composed of type I collagen, but the rupture areas express more type III collagen, which is thinner and withstands loads less effectively. Type III collagen accumulates slowly in the tendon, since its production does not increase very much, a situation that is indicative of microtrauma. Crosslinking of the fibres is important for collagen matrix properties, and we found that there is a change in the quality of crosslinking with age and that this may have role in the observed changes in tendon stiffness, as also noted in other studies. We also studied the appearance of tenascin-C at the rupture site in the Achilles tendon and at two other sites in the same tendon, but found no difference in its expression. It has been proposed that tenascin-C may take part in the tendon’s reaction to loading, but its exact function remains unknown.

Rheumatoid arthritis (RA) which affects 0.5-1% of the world population and is characterised by joint erosions and presence of the autoantibodies anti-citrullinated protein antibodies (ACPA) and rheumatoid factor. Collagen II (CII) is a joint-specific antigen and we have shown that antibodies against CII (anti-CII) are present in around 8% of RA patients. RA patients with anti-CII are characterized by acute RA onset with elevated CRP and early joint erosions at the time of RA onset. Polymorphonuclear granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) are abundant in RA synovial fluids, where they can interact with anti-CII, thus forming immune complexes (IC) with CII. In my thesis I have shown that PMN upregulated the cell surface markers CD66b and CD11b and downregulated CD16 and CD32 after stimulation with anti-CII IC. These changes in CD66b and CD16 associated to joint erosions to a larger extent than did PBMC responses to anti-CII IC. PMN cocultured with PBMC and stimulated with anti-CII IC showed augmented chemokine production that was dependent on TLR4 and functionally active PMN enzymes. This mechanism can lead to accumulation of inflammatory cells in joints of RA patients who are anti-CII positive around the time of RA diagnosis, and may thus help explain the acute onset RA phenotype associated with anti-CII. In a large Swedish RA cohort, anti-CII associated with elevations in clinical and laboratory measures of disease activity at diagnosis and until 6 months, whereas ACPA associated with late inflammation. Anti-CII seropositive RA was associated with improvements in clinical measurements and was negatively associated with smoking in contrast to ACPA that was associated with worseneing of clinical symptoms and associated positively with smoking. Anti-CII levels associated to  HLADRB1*03 and  HLADRB1*01 whereas ACPA showed negative association to HLA-DRB1*03. In a Malaysian RA cohort anti-CII also associated to elevated CRP at the time of diagnosis. Anti-CII seropositive RA represents a distinct phenotype, in many respects representing the converse  to the clinical, genetic and smoking associations described for ACPA. Early determinations of anti-CII in parallel to ACPA predict the inflammatory outcome in RA.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour in adults. The employment of current standard of care management strategy, that is combining maximum but safe surgical resection, and concomitant chemoradiotherapy, only achieves very modest survival benefits. Antiangiogenesis is a widely studied therapeutic strategy, which restricts the tumour growth by cutting off blood supplement. Although several antiangiogenic agents are now under clinicaland preclinical trials, bevacizumab is still the only one that has been proven to be effective in the treatment of recurrent GBM. However, the clinical use of bevacizumab has encountered the emergence of drug resistance. Its therapeutic benefit is considered limited because of its single pathway targeting. Many researchers believe that the use of broad spectrum angiogenesis inhibitors may leadto better clinical outcomes by overcoming the shortcomings of bevacizumab. / Vastatin, the globular non-collagenous 1 (NC1) domain of collagen VIIIα1, was initially proved to inhibit the proliferation and migration of bovine aortic endothelial cells. Although vastatin is similar in origin to other collagen-derived antiangiogenic factors (CDAFs), its antiangiogenic capability in treatment of cancers has not been studied systematically. Our team members previously found that vastatin wasa safe and effective antiangiogenic therapeutic and a potential biomarker for liver cancer. In this thesis, I tried to explore the therapeutic potential of vastatin in treatment of GBM. / Using a recombinant adeno-associated virus mediated gene therapy, the antiangiogenic potential of vastatin was first confirmed in vitro that it inhibited proliferation, migration and tube formation of murine microvascular endothelial cells (MECs). These effects were further confirmed using another gene vector (H1) which was subsequently employed for the in vivo studies. H1 is a nanopolymer gene vector has high affinity with the folate receptors on tumour cells. Transfection ofH1/vastatin reduced MEC proliferation in a U87/MEC co-culture system, suggesting a paracrine inhibition. Mechanism studies showed that vastatin caused a wide range of changes in the global gene transcription level in MECs, indicating a broad spectrum of action. / Following the establishment of an orthotopic murine GBM model, the H1/DNA polyplexes were injected directly to the tumour area. Treatment induced a significant increase in intracranial mRNA level of the therapeutic gene. Both vastatin and endostatin, a positive control, prolonged the survivals of GBM bearing mice. Immunostaning showed that vastatin decreased microvessel density in the outer layer of the tumour, while decreased cell density and caused abnormal vessel structures inthe centre. No synergistic effect was observed when GBM was treated with the combination of H1/vastatin and temozolomide (TMZ) in this model. / Finally, the therapeutic effect of vastatin on a TMZ resistant model was studied. GBM cells with acquired TMZ resistance (ATR) were established by chronic exposure of U87 cells to TMZ. Animals grafted with the U87-ATR cells were proved to be tolerant of TMZ treatment. H1/vastatin injection significantly prolonged the survival in this model. More interestingly, H1/vastatin also resensitized these animals to TMZ treatment. Stem cell related drug resistance was supposed to be disturbed in this process. / In conclusion, the present study has demonstrated for the first time that vastatin, a broad spectrum endogenous angiogenesis inhibitor, is of therapeutic benefit in a murine orthotopic GBM model. Vastatin’s capability to reverse TMZ resistance highlights an important area for further research. / 胶质母细胞瘤(GBM)是成人最常见的恶性原发性脑肿瘤。目前的治疗手段包括了手术切除和放化疗，但是效果仍不能让人满意。与传统的化疗药不同，抗血管生成药物能通过抑制肿瘤内新血管的形成，切断血流供给，达到限制肿瘤生长的目标。贝伐单抗（Bevacizumab)是目前唯一获得批准用于临床GBM治疗的抗血管生成药物。然而Bevacizumab在临床应用中必须面对耐药性产生的问题， 而且因为Bevacizumab只单一性地阻断血管内皮生长因子相关的通路，所以它的治疗效果也受到了一定程度的限制，让肿瘤可以选择替代性的通路来获得新生血管。因此一些研究人员认为，改用多靶点或者广谱的抗血管生成药物，治疗效果应该会更好。 / Vastatin是VIII型胶原蛋白α1链上的球状非胶原裂解片断。人体内这一类的片段多被证明了具有抗血管生成的功能，它们统称为“源自胶原蛋白的抗血管生成因子”。Vastatin具有抑制牛主动脉内皮细胞增殖和迁移的作用，然而它在抗肿瘤血管生成方面的作用却没有被系统地研究过。我们之前的实验曾经发现Vastatin对肝癌模型中的血管生成具有明显的抑制效果，而本论文将对Vastatin是否同样具有治疗GBM的作用展开研究。 / 在体外，我们首先证明了重组腺相关病毒（rAAV）介导的Vastatin基因治疗能有效抑制MEC的增殖和迁移，并阻止其形成管状结构。我们同时也测试了另一种基因载体H1，以方便后续动物实验的开展。H1是一种纳米聚合物，对肿瘤细胞表面高表达的叶酸受体有高亲和力。H1 介导的Vastatin 基因治疗对肿瘤细胞和MEC都没有直接的作用，但在两种细胞的共培养体系中，Vastatin可以通过旁分泌的方式来抑制MEC的增殖。对机制的研究发现，Vastatin使MEC内基因转录的水平发生了大范围多通路的改变，说明了它的作用具有一定的广谱性。 / 实验进一步研究了Vastatin在小鼠原位GBM 模型中的作用。将H1/DNA 复合物直接注入瘤区可以明显提高颅内相应基因的转录水平。Vastatin和作为阳性对照的Endostatin都能有效地延长GBM小鼠的生存期。免疫组织化学的结果显示Vastatin 能降低肿瘤内部的微血管密度，并诱导组织坏死。这与之前报道过的Endostatin的作用相似。在同一模型上，我们还测试了Vastatin和Temozolomide(TMZ)结合给药的效果，但并没有了现明显的协同作用。 / 实验最后研究了Vastatin在TMZ耐药模型中的治疗效果。通过将U87细胞长期浸泡中含有TMZ的培养基中，我们成功地筛选出了具有TMZ耐药性的GBM细胞。用这些细胞建立的小鼠GBM模型对TMZ的作用不敏感。实验表明，H1/Vastatin基因疗法不仅能够明显延长模型小鼠的生存期，还可以逆转耐药性，使TMZ重新发挥作用。我们推测干细胞相关的耐药性的产生和维持可能在这个过程中受到了影响。 / 上述研究第一次阐明了Vastatin对GBM的治疗效果。Vastatin具有广谱的抗血管特性，能够通过作用于MEC抑制肿瘤内部新血管的生成。Vastatin不仅本身具有治疗作用，还能逆转动物模型对化疗药物的耐受性，因些具有很高的研究价值。相信对Vastatin更一步的探索不但可以拓宽我们对抗血管生成药物的理解，也可能意味着一个新的研究领域的出现。 / Li, Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 102-110). / Abstracts also in Chinese. / Title from PDF title page (viewed on 05, January, 2017). / Li, Yi. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Abstract   Introduction Failure in the mechanism of self-tolerance in T or B cells can lead to autoimmunity. One of the autoimmune diseases is rheumatoid arthritis (RA), which is a chronic inflammatory disease of unknown cause and is characterized by systemic inflammation, autoantibodies and joint destruction. Serology is crucial for the classification of this disease. The first autoantibody found in RA patients was Rheumatoid factor (RF). However, anti-citrullinated peptide antibodies (ACPAs), a relatively new group of autoantibodies found in 70-90% of RA patients, are diagnostically more specific than RF. Type II collagen (CII) is the most abundant protein in human cartilage. In RA patients, immunity against CII leads to cartilage degradation and loss of joint function. Already from the 1970s, antibodies to CII (anti-CII) were found in RA sera, suggesting that CII autoimmunity might be pathogenetically important. Previous studies from our group show that a subgroup of patients with high levels of anti-CII at the time of diagnosis at the same time have high levels of inflammation in the joints. This is probably caused by anti-CII immune complexes (IC) inducing pro-inflammatory cytokines from macrophages. Although anti-CII positive patients have high inflammatory activity early on, as anti-CII levels decrease during the first year, the associated inflammation also diminishes. Thus, anti-CII positive patients have a rather good prognosis. Moreover, it is assumed that since anti-CII positive patients have a better prognosis than ACPA positive, patients with elevated anti-CII at the time of diagnosis might benefit from different and milder treatment. Previous studies from the group were performed on stored patient samples from the time before modern treatments with biologic agents (1995-2005). In this study, we aimed to investigate patients belonging to a more recent RA cohort, diagnosed between 2005-2014, with the aim to investigate whether patients with the anti-CII-associated RA phenotype would respond differently depending on the use of different modern RA therapies. Patients and Methods The primary cohort consisted of 2335 RA patients and 480 non-RA controls from the Epidermiological Investigations in Rheumatoid Arthritis (EIRA) case-control study. As we run into methodological problems two subgroups with 62 and 40 RA patients from the previous anti-CII studies were investigated when modifying the ELISA procedure, as well as a group of earlier investigated patients with non-specific ELISA reactivity. Totally 2776 RA patients were investigated. All investigated patients fulfilled the American College of Rheumatology classification criteria. To measure the anti-CII levels in RA patients and healthy controls anti-CII ELISA was performed. During the experiments, several different sources of CII from human, rat and bovine origin, and two different alternative coating buffers were used. The optical density (OD) was measured at 450 nm and anti-CII concentrations were calculated against the standard curve from an RA patient with high anti-CII levels.   Results My first analysis of the EIRA cohort showed that anti-CII are higher in RA patients than in controls, but could not confirm the association with acute onset RA. This was an unexpected finding and changed the focus of this master thesis project, to modify the measurement of anti-CII. Re-investigation of EIRA I showed that a proprietary coating buffer is important in the assay. Moreover, when different samples from RA patients were tested with bovine, rat and three different lots of human CII, correlation tests with clinical measures showed that bovine collagen and a new lot of human CII- prepared by the supplying company solely for this project- showed the strongest associations. Thereafter the EIRA cohort was re-investigated with two ELISAs, using bovine and human CII coated with the proprietary buffer. At the time of thesis writing almost all of the EIRA samples have been re-analysed, and results from both the modified ELISAs show the awaited clinical associations to early inflammation.   Conclusion Keeping the integrity of triple helical collagen is very important for the identification anti-native CII in RA patients. Our results show that the use of the proprietary coating buffer appears to be instrumental in this assay, irrespective of what source of CII was used. The new lot of human CII shows significant associations with the clinical measures, but associations are somewhat stronger with bovine CII. After finalising the re-investigations, we will be able to conclude which of the two analyses is most appropriate, and the corresponding dataset will then be merged with data from the first part of the EIRA study investigated previously by other group members. As anti-CII analysis shows the association to disease activity and prognosis, it can be used for predicting prognosis of RA and choosing the appropriate therapy in newly diagnosed RA patients, which might be clinically useful for rheumatologists. Our hypothesis is that as anti-CII positive patients have strong early inflammatory response but good long-term prognosis, they might benefit from other and perhaps short-term treatment compared to other RA patients.  If this is correct, our finding can have impact on the economy as it can define the patients who will not need expensive long-term medications. As modern anti-rheumatic therapies carry the risk of infections, such individualized therapies might also benefit anti-CII positive patients.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Abstract In this work the type XVIII collagen has been studied by using several approaches, such as different animal models. The primary structure of frog, Xenopus laevis, type XVIII collagen and the expression pattern of its variants during early embryogenesis have been elucidated. The gene structure of human type XVIII collagen was characterized and the localization and processing of its longest variant was studied by generated antibodies. In addition, the function of the proteolytically released C-terminal part of type XVIII collagen, endostatin, was studied by generating transgenic mice expressing endostatin. The primary structure of X. laevis type XVIII collagen is comprised of three N-terminal variants resembling their mammalian counterparts. The sizes of the polypeptides are 1285, 1581, and 1886 residues. The most conserved regions are the C-terminal endostatin region and the cysteine-rich domain in the N-terminus. Whole-mount in situ hybridization reveals different expression patterns for variants during embryogenesis. The short variant is the most abundant, whereas the two longest variants exhibit more restricted expression. The gene structure of human type XVIII collagen reveals an exon-intron organization that is conserved with mouse. The length of the human gene is about 105 kb and contains 43 exons. The third variant of type XVIII collagen has a conserved cysteine-rich domain with homology to the extracellular part of frizzled proteins. This third variant is localized to developing muscle and lung, and is also found in serum. In cell culture, the proteolytic fragments of the N-terminus, including the cysteine-rich motif, are also detected. Endostatin function was studied by generating mouse lines expressing endostatin under the keratin-14 promoter, which drives the expression mainly in the skin. Three independent transgenic mouse lines were achieved with varied expression levels. The phenotype was seen in the eye with lens opacity and abnormal morphology of epithelial cells in the lens. In the skin, a broading of the basement membrane in the epidermis dermis junction was detected. Immunoelectron microscopy analysis revealed a polarized orientation of type XVIII collagen in the basement membrane. In transgenic mice, altered localization of endogenous type XVIII collagen was seen, suggesting displacement of the endogenous type XVIII collagen with transgenic endostatin leading to disorganized basement membrane.

Abstract Collagen IX belongs to the superfamily of collagenous proteins and is present on the surface of the heterotypic collagen fibrils that are predominantly composed of collagen II, and also collagen XI. The major sites of expression of collagen IX include the articular cartilage, intervertebral disc, inner ear and the vitreous body of the eye. Previous reports have indicated that mutations in the genes encoding the three polypeptide chains of collagen IX may lead to intervertebral disc disease and multiple epiphyseal dysplasia, a chondrodysplasia characterized by early onset osteoarthritis. These observations and results from genetically modified mouse lines suggest that collagen IX is crucial in the maintenance of the long-term integrity of tissues. However, the structure-function relationship as well as detailed information concerning the functional roles of this protein has remained unclear. Recombinant human collagen IX was obtained using an insect cell expression system. Besides full-length molecules, five truncated variants of collagen IX were produced to examine chain association and trimerization. Contrary to previous observations, it was shown that the COL1 and NC1 domains are not essential for trimerization. Instead, they seem to play an important role in the specificity of chain selection. The results also suggest that the N-terminal domains, NC3 or COL3, are required for complete folding and stabilization of collagen IX molecules, implicating cooperativity between different domains in the folding process. Collagen IX was found to mediate cell adhesion and bind efficiently to collagen receptor integrins α1β1, α2β1, α10β1 and α11β1. The binding was found to represent a novel type of mechanism, and the binding site of the integrin I domain was located at the N-terminal end of the COL3 domain in collagen IX. The obtained results suggest that the FACITs may play an important role as mediators of cell adhesion to collagen fibrils. Antibodies binding to human recombinant collagen IX were measured among 53 patients with seropositive rheumatoid arthritis (RA). These autoantibodies were significantly elevated among the RA patients when compared to the controls, suggesting that autoantibodies to collagen IX show diagnostic potential in early RA. However, no association was found between the antibody levels and outcome.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.