Дисертації з теми "3D skin infection model"

Psoriasiform skin diseases are a poorly understood group of disorders. Recent data has implicated the immune system with a central role in disease pathogenesis. In this thesis, various T cell populations were studied in a TCR transgenic model of psoriasiform disease, whose abnormal interactions culminate in distinctive psoriasiform pathology. The model is based upon the expression of the transgenic 2C TCR on the H-2[sup d] -expressing DBA/2 inbred strain (referred to as D2C mice). The 2C TCR recognizes a peptide (p2Ca) derived from the ubiquitous mitochondrial protein 2-oxoglutarate dehydrogenase, presented by the MHC class I molecule, L[sup d]. In D2C mice, expression of the 2C TCR led to a comprehensive depletion of immature T cell progenitors, which express the 2C TCR, and a resultant lymphopenia of mature peripheral T cells. The lymphopenia of CD8⁺ cytotoxic and CD4⁺ helper T cells predisposes to the overgrowth of opportunistic pathogens, resulting in inflammatory skin disease restricted to the sebum rich areas of the skin, resembling the human psoriasiform disease seborrheic dermatitis. In D2C mice, there is also a deficiency in T regulatory (T[sub reg]) cells as a result of slowed thymic output of mature T cells. The reduced T[sub reg] function results in a lymphoproliferation of polyclonal CD4⁺CD25⁻ cells, many of which home to the aforementioned cutaneous inflammatory sites. This expansion of "helper" cells was likely due to antigenic stimulation, presumably against immunogenic determinants of opportunistic pathogens. Reconstitution of the T[sub reg] compartment by adoptive transfer abrogates the development of psoriasiform pathology and precludes the lymphoproliferation of CD⁺CD25⁻ cells. These data suggest that T[sub reg] may downregulate the response of mature cells to ubiquitous commensal organisms as a means to maintain immunological homeostasis. In TCR transgenic mice which express the cognate Ag of the transgenic TCR, a large population of transgenic cells exist in the peripheral lymphoid tissues. These cells are anergized and fail to respond to stimulation with cognate ligand; however, they express a memory immunophenotype, including the intermediate affinity IL-2 receptor. The expression of this receptor enables these cells to use bystander IL-2 or IL-15 to overcome this inactivation, revealing enhanced functional properties induced by the high-affinity interaction with cognate ligand. These observations suggest that such clonally anergized cells may represent an in vivo autoimmune hazard; and, interestingly, a further consequence of the CD4⁺CD25⁻ lymphoproliferation in D2C mice is the bystander activation of these transgenic T cells. After undergoing acute activation, 2C T cells exacerbate the cutaneous pathology in these animals, a consequence that can be abrogated by the administration of a blocking mAb against the 2C TCR. Interestingly, the combination of immunodeficiency, T[sub reg] lymphopenia, the presence of CD4⁺CD25⁻ cells capable of undergoing vigorous expansion, and a large population of memory phenotype 2C transgenic cells was insufficient to induce disease when D2C bone marrow was adoptively transferred to lethally irradiated syngeneic DBA/2 mice. Examination of these animals revealed that bone marrow transfer did not deplete the skin of DBA/2-derived cutaneous γδ cells. Sentinel intraepithelial γδ lymphocytes have been shown to have an important role in surveying the epithelium for signs of infection and malignancy as well as in maintaining epithelial integrity. The development of these cells is curtailed in D2C mice due to the forced expression of the 2C TCR; however, the persistence of these cells in the aforementioned bone marrow chimeras may have protected these animals from the development of the disease phenotype. While generated DBA/2 TCRδ⁻/⁻ mice did not develop spontaneous disease, the transfer of D2C bone marrow to lethally irradiated DBA/2 TCRδ⁻/⁻ recipients successfully transferred the disease phenotype, confirming the importance of these cells in protecting against the development of psoriasiform pathology. This result also demonstrated that a compromised cutaneous barrier is necessary for disease pathogenesis, as disease does not develop when the skin is populated by sentinel intraepithelial lymphocytes. While considerable research efforts have been focused on human psoriasiform disease, a solid understanding of disease pathophysiology is severely lacking. This limited knowledge of psoriasiform disease is highlighted by the ongoing uncertainty of whether these diseases represent primary diseases of the epithelium, or whether these diseases represent tissue specific autoimmunity occurring in normal skin as a result of dysfunctional immunity. One explanation for this failure to understand basic principles of psoriasiform disease pathophysiology can be attributed to the limited numbers of appropriate model systems to carefully study disease. The D2C model of psoriasiform disease has been shown to be an accurate model system which has demonstrated that a complex interplay between various immunocytes and epithelium culminates in psoriasiform disease. The insight that the D2C model has generated will lead to a better understanding of these poorly characterized psoriasiform conditions.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Skin is an organ with a complex structure which plays a crucial role in thebody’s defence against external threats and in maintaining major homeostatic functions. The need for in vitro models that mimic the in vivo milieu is therefore high and relevant with various applications including, among others, penetration, absorption, and toxicity studies. In this context, the choice of the biomaterial that will provide a 3D scaffold to the cultured cells is defining the model’s success. The FN-4RepCT silk is here suggested as a potent biomaterial for skin tissue engineering applications. This recombinantly produced spider silk protein (FN-4RepCT), which can self-assemble into fibrils, creates a robust and elastic matrice with high bioactivity, due to its functionalization with the fibronectin derived RGD-containing peptide. Hence it overcomes the drawbacks of other available biomaterials either synthetic or based on animal derived proteins. Additionally, the FN-4RepCT silk protein can be cast in various 3D formats, two of which are utilized within this project. We herein present a bilayered skin tissue equivalent supported by the FN-4RepCT silk. This is constructed by the combination of a foam format, integrated with dermal fibroblasts and endothelial cells, and a membrane format supporting epidermal keratinocytes. As a result, a vascularized dermal layer that contains ECM components (Collagen I, Collagen III, and Elastin) is constructed and attached to an epidermal layer of differentiated keratinocytes.The protocol presented in this project offers a successful method of evenly integrating cells in the FN-4RepCT silk scaffold, while preserving their ability to resume some of their major in vivo functions like proliferation, ECM secretion, construction of vascular networks, and differentiation. The obtained results were evaluated with immunofluorescence stainings of various markers of interest and further analysed, when necessary, with image processing tools. The results that ensued from the herein presented protocol strongly suggest that the FN-4RepCT silk is a promising biomaterial for skin tissue engineering applications.

Melanoma is a fatal form of skin cancer which progresses in an orchestrated pattern in human skin. Characterising these phases of melanoma in vitro can provide key insights into mechanisms of the disease progression. In this thesis, we investigate how in vitro three-dimensional (3D) model assays that recapitulate human skin can be used to identify key features underlying melanoma progression. In particular, we construct a 3D melanoma skin equivalent model using melanoma cells from the early and late phase of the disease. We further quantify melanoma cell migration, proliferation, invasion, as well as melanoma nest formation.

Background: Necatoriasis is a neglected tropical disease caused by the insidious parasite Necator americanus. This hookworm infects and reinfects approximately 500 million individuals worldwide, with a further 5.1 billion at high risk for acquiring the infection. Despite the high level of reinfection, no lasting immunological memory develops in the host. Albeit the profound health implications, chronicity and public health burden in developing countries, many aspects of human Necator americanus infection, particularly early events at the interface with the host immune system, are under researched. These figures and facts highlight the need for new research elucidating the molecular interactions between Necator americanus and the innate immune system. This will aid in the rational design of innovative and more efficient intervention strategies against hookworm infection, which is an essential measure for disease prevention. Objectives: In the context of Necatoriasis, this thesis studied the physical interaction between infective Necator americanus larvae (L3) with human dendritic cells (DCs) and epidermal keratinocytes, investigating the biological consequences. In addition, the development of a platform consisting of human keratinocytes, fibroblast and DCs on a 3D scaffold was constructed as an in vitro model of human skin. Results: The present thesis provides new insights into early immunological events at the interface of DCs and Necator americanus larvae and could explain how L3 affect immunity upon initial interaction with antigen presenting cells. For the first time, the data presented illustrates the sequestration of human DCs onto the sheath of L3 infective Necator americanus larvae, triggering the hookworm to exsheath. Intriguingly, the exposed cuticle of the larvae had negligible interaction with the free DCs. The findings also illustrate that the interaction between DCs and the larvae is mediated via a mandatory interaction with C-type lectin receptors, dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose receptor (MR). Blocking of either receptors with antibodies resulted in an inhibition of DC sequestration and aggregate formation. This demonstrates the biological relevance of previously identified lectin binding molecules on the Necator americanus larvae (L3) sheath in the context of interacting with DCs. These findings allude to a disparity between the surface chemistry of the sheath and larvae that could explain their differential ability to interact with DCs. While the exact nature of differences in the surface properties of the larvae and sheath are yet to be characterised, this data clearly indicates the presence of distinct chemical signatures on the cuticle sheath that attract DCs. However, this not only induces exsheathing but also enables larvae migration without being recognised or challenged by antigen presenting cells. A potential escape mechanism through which the larvae could bypass the immune cells, creating a possible site of ‘temporary immune privilege’. DCs incubated with viable axenic larvae exhibited an immature phenotype as evidenced by the low expression of the maturation markers CD80, CD83, CD86, CD40, and HLA-DR. Subsequently, the ability of DCs to acquire a mature phenotype in response to co-stimulation with lipopolysaccharide (LPS) in the presence of Necator americanus was assessed. These data show that DCs treated with the larvae will remain responsive to LPS stimulation. Additionally while the axenised larvae do not induce any cytokine production by DCs, they seem to suppress LPS induced cytokine expression, however these changes were not statistically significant (p value ≤0.3). Furthermore, the cell-free culture media from DCs, matured in the presence of LPS, had no visible effects on the larvae. Intriguingly, matured DCs in LPS-free culture media render the larvae non-viable through a lysing mechanism, alluding to a modified paracrine signalling response by mature immune cells in culture with the parasite. Interestingly, in the presence of epidermal keratinocytes, ex-sheathing was not mandatory to enable larval burrowing. In fact, only a small number of the larvae sheaths were recoverable from the apical surface of the keratinocyte layer; indicating preferential ensheathed larval burrowing. The data also illustrated the novel behavioural strategies promoting host invasion by Necator americanus larvae, in the presence of epidermal keratinocytes. Larvae were notably slower to exsheath in culture with keratinocytes and exhibited no vigorous movements as observed in DC cultures. This was thought to prevent early exsheathing, as the advantage of larvae maintaining their sheath during the initial stages of infection is in theory highly beneficial. Finally an immunocompetent tri-culture was developed on 3D layered PET scaffolds, encompassing epidermal keratinocytes and dermal fibroblasts, interspersed with DCs cultured at air liquid interface. A functional barrier was optimised, following which immune cell migration within the tri-culture system was observed successfully. Conclusion: Collectively, the sequestration of DCs onto the larvae sheath, suppression of maturation and cytokine expression, provides a possible explanation for the lack of a lasting immune response. These data provide novel insights into early immunological events at the interface of DCs, epidermal keratinocytes and Necator americanus larvae, which could explain how L3 evade immunity upon initial interaction with antigen presenting cells.

La détection de la protéine prion anormale dans les tissus lymphoïdes de patients britanniques suggère qu’après exposition à l’agent de la variante de la maladie de Creutzfeldt-Jakob (vMCJ) plus de 99% des contaminations pourraient demeurer cliniquement silencieuses. Ces données soulignent un risque de transmission secondaire par transfusion sanguine ce qui nous a conduit à une étude expérimentale. En parallèle des formes classiques de vMCJ, nos modèles murins et simiens de retransmission ont mis en evidence des phenotypes atypiques. Ces phénotypes échappent actuellement aux critères de diagnostic puisqu’aucune protéine prion anormale (PrPres) n’est détectée.Nos travaux ont eu pour but principal d’évaluer expérimentalement le risque sanguin au travers d’études de retransmission et de caractérisation de la replication des souches classiques et atypiques aux niveaux périphérique et central.Nous observons une très forte hétérogénéité dans la réplication de la PrP anormale dans les différents tissus lymphoïdes des macaques transfusés développant une vMCJ. Le niveau de contamination des tissus lymphoïdes apparait proportionnel à l’infectiosité sanguine de ces animaux et au risque de transmission de la maladie in vivo. Concernant les formes atypiques, la majorité des macaques transfusés n’ont pas de réplication dans les tissus lymphoïdes bien que ces phénotypes soient transmissibles expérimentalement à des modèles murins. Des transmissions à des souris immunodéficientes révèlent que les souches atypiques sont transmissibles par voie périphérique en l’absence d’un système immunitaire fonctionnel.Une alternative à l’expérimentation animale a été réalisée grâce aux « mini-brains » mimant la complexité du cerveau humain. Ces organoïdes cultivés en trois dimensions sont sensibles à au moins un isolat de prion associé aux formes sporadiques humaines. Les mini-brains pourraient ainsi constituer un nouvel outil d’étude des maladies à prions et permettre à termes la caractérisation des souches atypiques
The detection of abnormal prion protein in the lymphoid tissues of UK patients suggests that after exposure to the agent of variant Creutzfeldt-Jakob disease (vCJD), more than 99% of contaminations may remain clinically silent. These data highlight a risk of secondary transmission through blood transfusion. In parallel to the classical vCJD forms, our experimental models in mice and macaques revealed another group which avoids the current diagnostic criteria, including the absence of abnormal prion protein (PrPres).The main goal of our work was to experimentally assess the risk of blood through retransmission studies and characterization of the abnormal replication of classical and atypical strains examined at peripheral and central levels.We observed a high heterogeneity of the distribution of the abnormal PrP in the lymphoid tissues of vCJD transfused macaques. The global level of contamination in lymphoid tissues seems proportional to the blood infectivity in these animals and to the risk of in vivo transmission of the disease. Regarding atypical forms, despite an absence of replication in lymphoid tissues, these phenotypes are experimentally transmissible. Transmissions to immunodeficient mice reveal that atypical strains are transmissible through peripheral routes in the absence of functional immune system.An alternative to animal testing has been achieved using to "mini-brains" mimicking the complexity of the human nervous system. These organoids cultured in three dimensions are sensitive to at least one prion isolate associated with human sporadic forms. Thus, mini-brains could constitute a new tool for studying prion diseases and improve the characterization of atypical strains

Includes bibliographical references
Burn injuries are among the most devastating of all injuries and a major global public health crisis, with fire related burns accounting for approximately 265 000 deaths annually. The African continent, most especially Sub-Saharan Africa, has the second highest mortality rates (15% of global mortality rates). In South Africa, 3.2 % of the total population sustains burn injuries, with 50 % of these cases as children under the age of20 years. Studies have also shown that most of these incidences are prevalent within the age groups of 0-5 years, and account for the 3rd most common cause of mortality in children under the age of 15 years. In depth knowledge and understanding of cellular facets of wound healing has allowed for a greater stance in the interventions aimed at circumventing problems associated with development of effective wound defects treatment regimen. Burn treatment options are largely dependent on the degree and extensiveness of burns. A wide body of literature exists with regards to traditional as well as current treatment options. These include, for instance the use of various forms of skin auto-grafts. Despite such great success with all kinds of innovative ideas surrounding the use of autologous skin grafting, lack of available donor sites for skin grafts still remains a problem, more so in cases where patients suffer burns spanning more than 70% TBSA. This therefore has inspired the design and use of bioengineered skin substitutes as well as cultured/non-cultured autologous epidermal cells. Unfortunately, to date, no tissue engineering technique has fully been able to recapitulate the anatomy and physiology of the skin, or has attained the biological stability as well as achieving the aesthetic outcome. Several hurdles are yet to be overcome to achieve this. Amongst many, inclusion of melanocytes, other skin appendages as well as potential progenitor cells is some of the attributes of an ideal 3D skin equivalent. Therefore pigmented 3D skin constructs are of great interest as they address not only the issues of complete wound healing, but also the aesthetic outcomes. In light of this, correct keratinocyte to melanocyte ratios are also of great importance in designing such pigmented 3D constructs. Therefore the major aim of this study was to isolate skin melanocytes and keratinocytes, and co-culture them at different ratios in order to attain optimal pigment production and/or consequent improved wound healing outcome. To determine the best keratinocyte to melanocyte ratio to use in developing pigmented3D skin constructs, the following co-culture ratios were used: 5:1, 10:1 and 20:1.Proliferation assays were employed to further elucidate the growth dynamics of both human skin melanocytes and keratinocytes in either mono- or co-culture system. Secondly, FACS was used to develop a reliable technique to be used to separate the two cell types from a co-culture system in order to perform downstream analyses. Thirdly, to establish the roles of the co-cultured cells in wound healing (with regards to proliferation and migration), scratch wound healing assays were employed. Lastly, FACS was used to infer the effect of such ratios on pigment production. Our results demonstrated that keratinocytes, compared to melanocytes mono-cultures have higher proliferation capacity. On the contrary melanocyte's proliferation is up-regulated by the presence of keratinocytes in a co-culture, whereas higher numbers of melanocytes in co-culture with keratinocytes resulted in less proliferative keratinocyte phenotype. The FACS separation technique worked excellently in identifying keratinocyte population from melanocytes, with an almost 100% accuracy. This is shown by melanocytes being sorted as 93% of MART-1 + cells in a mono-culture, followed by an approximately 5:1 separation of keratinocytes from melanocytes (77% Kc and 17% Mc). In vitro scratch assays demonstrated that none of the co-culture ratios was significantly superior with regards to wound healing capacities and pigment production, in the absence of fibroblast-conditioned medium. In conclusion, the 5:1 co-culture ratio seemed to yield a non-significant, yet best outcome with regards to wound healing capacity (only in the presence of fibroblast-derived factors), thus conferring it as a potential optimal ratio of keratinocytes to melanocytes, to be used in development of our pigmented 3D constructs.

This master’s thesis deals with the calculation of electrodynamic forces breaker BD250NE305. Main tasks in this semester project is to study the theoretical analysis of individual parts specified breakers. Processing theoretical analysis of these forces. Creating a 3D model current path and sheets quenching chamber single phase circuit breaker in Autodesk Inventor Professional 2012. Another challenge is the subsequent export the model into the simulation program ANSYS Maxwell. After simulation, the specified conditions must be processed and the results of the present work is to evaluate.

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Wound infection is a major clinical problem, yet understanding of bacterial host interactions in the skin remains limited. Microbe-derived molecules, known as pathogen-associated molecular patterns, are recognised in barrier tissues by pattern-recognition receptors. In particular, the pathogen-associated molecular pattern, lipopolysaccharide (LPS), a component of microbial cell walls and a specific ligand for Toll-like receptor 4, has been widely used to mimic systemic and local infection across a range of tissues. Here we administered LPS derived from Klebsiella pneumoniae, a species of bacteria that is emerging as a wound-associated pathogen, to full-thickness cutaneous wounds in C57/BL6 mice. Early in healing, LPS-treated wounds displayed increased local apoptosis and reduced proliferation. Subsequent healing progression was delayed with reduced re-epithelialisation, increased proliferation, a heightened inflammatory response and perturbed wound matrix deposition. Our group and others have previously demonstrated the beneficial effects of 17β-estradiol treatment across a range of preclinical wound models. Here we asked whether oestrogen would effectively promote healing in our LPS bacterial infection model. Intriguingly, co-treatment with 17β-estradiol was able to promote re-epithelialisation, dampen inflammation and induce collagen deposition in our LPS-delayed healing model. Collectively, these studies validate K. pneumoniae-derived LPS treatment as a simple yet effective model of bacterial wound infection, while providing the first indication that oestrogen could promote cutaneous healing in the presence of infection, further strengthening the case for its therapeutic use.

Wound infection is a major clinical burden that can significantly impede the healing process and cause severe pain. Prolonged wound infection can lead to long-term hospitalization or death. Pre-clinical research to evaluate new drugs or treatment strategies relies on animal studies. However, animal studies have several challenges including interspecies variations, cost, and, ethics question the success of these models. Recent advances in tissue engineering have enabled the development of in vitro human skin models for wound infection modeling and drug testing. The existing skin models are mostly representative of the healthy human skin and its normal functions. However, to study the wound healing process and the response of skin to the infection, there is still a need to develop a skin model mimicking the wound infection. This work presents a simplified functional infected epidermis model, fabricated with enzymatically crosslinked gelatin hydrogel. The immortalized human keratinocytes, HaCaT cells, was successfully cultured and differentiated to a multilayer epidermis structure at the air-liquid interface, and expressed terminal differentiation marker, filaggrin, in the outer layer. The barrier function of the epidermis model was studied by measuring the electrical resistance and tissue permeability across the layer. The results showed that the developed epidermis model offered a higher electrical resistance and a lower drug permeability compared to the cell monolayer on gelatin and cell-free gelatin. To show the capability of the developed epidermis model in wound modeling and drug, the model was infected with Escherichia coli and the inflammatory response of keratinocytes was studied by measuring the level of proinflammatory cytokines, including IL-1β and TNF-α. The results demonstrated the proinflammatory response of the epidermis model to infection by producing a higher level of TNF-α and IL-1β compared to the control group. While treating with antibiotic ciprofloxacin terminated the proinflammatory response and reduced the level of TNF-α and IL-1β. The robust fabrication procedure and functionality of this model suggest that this model has great potential for wound modeling and high throughput drug testing.
Graduate

Bovine Dermatitis digitalis (DD) ist eine weltweit verbreitete Infektionskrankheit bei Rindern, die primär die plantare Haut über dem Kronenrand nahe des Zwischenzehenspalts der Hinterklauen betrifft. Schmerzhafte ulzero-proliferative Läsionen mit akuten und chronischen Erscheinungsformen führen zu Verhaltensänderungen und Lahmheit der Tiere. DD hat damit einen erheblichen Einfluss auf deren Wohl und ihre Leistungen. Zahlreiche Untersuchungen zur Ätiologie der Krankheit ergaben, dass es sich um das Zusammenspiel verschiedener Ursachen handelt. Einer synergistischen multifaktoriellen Infektion mit starker Beteiligung von Bakterien der Gattung Treponema kommt dabei besondere Bedeutung zu. Aspekte wie Tierhaltung, Hygienestandards und genetische Prädispositionen wurden ebenfalls intensiv untersucht. Nichtsdestotrotz bleiben Infektionsherde, Transmissionsrouten und Pathomechanismen weitgehend unklar. Zum besseren Verständnis der Ereignisse, die zu DD-Läsionen führen, sollte im Zuge dieser Arbeit ein organotypisches Zellkulturmodell der bovinen Haut erstellt werden, welches in späteren Versuchen mit dem Krankheitserreger zum Einsatz kommen soll. Verlässliche und reproduzierbare Techniken zur Isolation und Kultur von bovinen primären Keratinozyten und Fibroblasten wurden etabliert; geeignete Zellkulturmedien für die Langzeitkultivierung und –aufbewahrung der Hautzellen wurden identifiziert. Zur Erstellung des Hautmodells wurden zwei verschiedene Ansätze miteinander verglichen. Der zweite Ansatz, bei dem Keratinozyten direkt auf ein dermales Äquivalent, d.h. ein Pad aus bovinem Kollagen I mit eingesäten post-mitotischen Fibroblasten, gesät wurden, brachte ein vielversprechendes Hautmodell hervor. Die inkorporierten post-mitotischen Fibroblasten wiesen eine charakteristische Zellmorphologie mit intakten Nuklei auf. Die terminale Differenzierung der Keratinozyten auf dem dermalen Äquivalent wurde mittels Immunfluoreszenzfärbungen mit Antikörpern gegen die Markerproteine Keratin 14 und Desmoglein 1 gezeigt. Die Ergebnisse erster Experimente mit Treponema spp. verdeutlichen, dass das Hautäquivalent ein geeignetes Modell zur Untersuchung der Pathogenese der DD darstellt.
Bovine digital dermatitis (DD) is a worldwide occurring, infectious disease in cattle primarily affecting the plantar skin above the coronary band near the interdigital cleft on hind feet. Painful ulceroproliferative lesions with acute and chronic appearances lead to behavioral changes and lameness. Hence, DD has a major impact on animal welfare and performance. Substantial efforts in investigating the etiology of the disease revealed a synergistic origin with evidence for a multibacterial infection and the strong involvement of bacteria from the genus Treponema. As the interaction between host, pathogen and environment is not negligible, surrounding circumstances such as housing, general hygiene and genetic predispositions have been investigated intensively. Nevertheless, infection reservoirs, transmission routes and pathomechanisms remain widely unclear. To better understand the cellular and molecular events during Treponema-infection of bovine skin, it was the specific aim of this study to establish an organotypic in vitro skin model, which could be challenged with the causative agent of the disease. A technique to reliably and reproducibly isolate primary keratinocytes and fibroblasts from the site of infection was established. Appropriate cell culture media for the long-term cultivation and storage of bovine skin cells were identified. Two different methods to develop the skin model were compared. The second strategy in which keratinocytes were directly seeded on top of a dermal equivalent, i.e. a bovine collagen type I pad with embedded post-mitotic fibroblasts, gave rise to a promising organotypic skin equivalent. The incorporated post-mitotic fibroblasts showed a characteristic cell morphology with intact nuclei. The terminal differentiation of the keratinocytes on top of the dermal equivalent was shown with anti-K14 and anti-Dsg1 immunofluorescence stainings. The results of initial Treponema-experiments proved that the skin equivalent is a suitable model to investigate the underlying mechanisms during Treponema-infection of bovine skin and hence, the pathogenesis of DD.

碩士
臺灣大學
免疫學研究所
98
Interleukin-15 (IL-15) is a pleiotropic cytokine that plays an important role in mediating innate and adaptive immunity in the host. The pedigree 191 (P191) of the ENU-mutagenized mice, generated by the Mouse Mutagenesis Program Core Facility (MMPCF) has been identified and predominantly express an alternatively spliced IL-15 mRNA called IL-15 ∆E7. Infection of P191 mutant mice via flank skin with herpes simplex virus-1 (HSV-1) showed a much more severely disrupted lesional skin than in B6 wildtype mice accompanied with enhanced HSV viral protein expression as well as elevated expressions of IL-1β and IL-6 in P191 lesional skin by cDNA microarray and real-time PCR analysis. How the function of IL-15 is affected and/or regulated by IL-15 ∆E7 and thus results in the altered inflammatory response against HSV-1 infection will be further investigated. Consistent with the depressed CD44 expression on CD8+ T cells in P191, we also found that the percentages of CD8+ T cells from lymph nodes, spleen and peripheral blood expressing CD44hiCD122hi were reduced in P191 mice by flow cytometric analysis. Using Kb-HSV-gB498-505 tetramer reagent, we found that gB-specific CD8+ T cells were generated in a delayed kinetics in P191 as compared to wildtype mice. However, these gB-specific CD8+ T cells significantly expanded in the spleen of P191 on day 10 after infection and the absolute numbers of gB-specific CD8+ T cells were higher than these in B6 mice, indicating that these cells efficiently proliferated in P191 spleen on early times of infection. In proliferation experiment, CFSE-labeled T cells were stimulated with gB498-505 peptide in rIL-2 and rIL-15. Whereas HSV-primed CD8+ T cells from B6 mice proliferated to gB antigen in vitro, proliferation of HSV-primed CD8+ T cells from P191 was significantly reduced given with sufficient IL-15. This suggested that gB-specific CD8+ T cells generated in P191 were poorly responsive to recall antigen. Using FlowCytomix to profile cytokine expression in B6 and P191 mice after HSV-1 infection, we have found that the level of IFN-γ in spleen was significantly reduced in P191 as compared to B6 spleen. How the reduced production of IFN-γ is associated with less proliferation of antigen-specific CD8+ T cells from P191 requires further investigation. Results from current experiments have shown that inflammatory response in skin and the properties of antigen-specific CD8+ T cells induced by HSV-1 infection are both altered in P191. How expression of IL-15 splice variant is involved in the control of these phenotypes remains to be clarified.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021
The skin is a complex organ mainly responsible for protecting the body from external threats and maintaining homeostasis. It is a complex three-dimensional structure that is composed of two main compartments, the dermis and the epidermis. Due to increasing ethical and legal pressure on animal usage in research, reconstructed 3D human skin models have been gaining popularity. These models mimic human skin architecture in vitro and allow relatively easy manipulation to meet specific needs. Some rare diseases remain poorly studied and could take advantage of this technology. One example is the Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) which is an early-onset neurological disease that was first described in Quebec, Canada, but cases have been reported worldwide. Patients suffer from spasticity and lack of coordination of muscle movements, resulting in an early wheelchair dependence and premature death. ARSACS is caused by loss-of-function mutations in the SACS gene, leading to a defective sacsin protein. Sacsin loss of function has been linked to mitochondrial dysfunction and abnormalities in the organization of intermediate filaments, but the complete picture is still unclear. Evidence of abnormalities in the skin of ARSACS patients has been reported, making this disease an interesting candidate to be studied using in vitro skin models. In this work, two different human keratinocyte cell lines (HaCaT and N/TERT-1) were used to create new human epidermal models using a polycarbonate inert matrix. The localization of different keratins and other markers (keratins 10, 14 and 15, and involucrin) were studied to characterize epidermal differentiation and stratification. Sacsin expression was analyzed in different cell lines and sacsin knockdown was attempted in HaCaT keratinocytes using lentiviral shRNAs. The HaCaT cell line was unable to recreate the normal multi-layer architecture of native skin nor the stratum corneum. This cell line expressed low amounts of the sacsin protein, and no difference was observed between the knockdown and the control by western blot. N/TERT-1 keratinocytes generated a stratified epidermis with all the normal layers present, including the stratum corneum. Complete epidermal differentiation was confirmed by the differential expression of epidermal markers. K14 expression was limited to the basal layer, while K10 was expressed in the upper layers, as expected. Involucrin was mostly expressed in the stratum granulosum and K15 expression was overall very low, indicating a successful differentiation. Sacsin expression was verified in different skin cells (HEKn, HDFn, and N/TERT-1), and N/TERT-1 expressed sacsin in amounts slightly lower than primary human keratinocytes. These findings suggest that the N/TERT-1 cell line has more potential to produce an epidermal skin model with an ARSACS phenotype, which can prove an important tool in future research. Despite the existing knowledge about sacsin structure and function, a lot is still unknown about this protein and how it causes the symptoms underlying ARSACS disease. Advances in this topic could contribute to the development of therapies that could cure or tackle some of ARSACS symptoms to ensure a better quality of life for the patients.

碩士
國立臺灣大學
流行病學研究所
89
Background: The complicated disease natural history of tuberculosis in relation to tuberculosis skin test (TST) with the treatment of latent tuberculosis infection (LTBI) given the high coverage rate of BCG vaccination has not been fully addressed. Objective: The aims of this study are to assess the relative contributions of exogenous disease due to re-infection and endogenous and to perform a cost-effectiveness analysis for the use of TST plus LTBI by different screening regimes given the current BCG vaccination in the general population and in the high-risk group. Methods: A computer simulation model using the Markov model was proposed to model the disease natural history of TB. Markov cycle tree and Monte Carol simulation were employed to assess the relative contributions of exogenous disease, re-infection and endogenous reactivation. Cost-effectiveness analysis was performed to calculate the incremental cost-effectiveness ratio for averting TB cases and deaths by different screening regimes starting from 20, 30, 40, 50, and 60 years of age. Results: The incremental cost-effectiveness ratios to achieve an additional life-year in the screening regimes starting from 20, 30, 40, 50 and 60 years of age against no screening are calculated as $6410, $4072, $2858, $3863 and $13416, respectively, for the general population and $2183, $1506, $1126, $1567and $4913, respectively, for the high-risk group. The sensitivity analysis shows if the effectiveness of INH is dropped to 30% TST screening is no longer cost-effective. Conclusion: the present study develops a computer simulation model for elucidating the disease natural history of TB and cost-effectiveness analysis for TST with the treatment of LTBI. The results suggest that TST screening regime may be the most cost-effective at the start of 40 years of age for the selected high-risk group.