Rozprawy doktorskie na temat „Immunohistochemistry”

The aim of this project was to compare and contrast the results of three related methodologies which are of particular relevance or potential within our department -. a manually performed streptAvidin Biotin Complex (sABC/ HRP) method which for many years has served as our 'standard' immunocytochemical method; an integrated semi-automated systems from DAKO based on the TechMate 500 immunostainer and the ChemMate reagent package;. a manually performed tyramide amplification method regarded as amongst the most sensitive immunocytochemical method currently available. Antigen retrieval investigating differing durations of exposure to the proteolytic enzyme trypsin, and heat mediated antigen retrieval using citrate buffer pH6.0 or DAKO high pH antigen retrieval solution were also included as parameters for investigation. To this aim a panel of eleven specific antibodies were assessed, each of which had previously displayed individual qualities worthy of investigation -. PGP9.5, CD30, oestrogen receptor, CD3 polyclonal, CD2, CD3 monoclonal, CD4, cyclin D1, kappa immunoglobulin light chain, lambda immunoglobulin light chain and IgD immunoglobulin heavy chain. The results of this work demonstrate that each of the systems investigated has its individual merits -. The manual sABC/ HRP method is the least complicated and expensive of the methods to perform. There are significant disadvantages in its used however. Being a manual method there are many steps where individual variation in working practices within a laboratory can and do affect results. The TechMate/ ChemMate system is a more sensitive system than the manual sABC/ HRP method also providing a cleaner end result and allowing higher dilutions of primary antibody to be employed. Tyramide amplification is the most sensitive system evaluated, the antibodies in this work generally demonstrated immunostaining at higher dilutions than the other methods tested although some, in particular those directed against immunoglobulin light chains, operated better under the ChemMate/ TechMate system. On occasion specific positive results were obtained which none of the other methods tested could achieve with certain antibodies, CD3 monoclonal and to a lesser extent CD2.

This thesis focus on the analysis of digitized microscopic image, especially on IHC stained colour images. The corresponding contributions focused on the automatic detection of stain colour and glands, the segmentation and quantification of cell nuclei, the analysis of liver cirrhosis and the development of a semi-automatic toolbox. Colour is the most important feature in the analysis of immunostained images. We developed a statistical colour detection model for stain colour detection based on the histograms of collected colour pixels. This is acting on the approach "what you see is what you get" which outperforms the other methods on the detection of several kinds of stain colour. Verifying the presence of nuclei and quantifying positive nuclei is the foundation of cancer grading. We developed a novel seeded nuclei segmentation method which greatly improves the segmentation accuracy and reduces both over-segmentation and under-segmentation. This method has been demonstrated to be robust and accurate in both segmentation and quantification against manual labelling and counting in the evaluation process. The analysis of gland architecture, which reflects the cancer stage, has evolved into an important aspect of cancer detection. A novel morphology-based approach has been developed to segment gland structures in H-DAB stained images. This method locates the gland by focusing on its morphology and intensity characteristics, which covers variations in stain colours in different IHC images. The evaluation results have demonstrated the improvements of accuracy and efficiency. For the successive development of three methods, we put them in a semi-automatic toolbox for the aid of IHC image analysis. It can detect different kinds of stain colour and the basic components in an IHC image. The user created models and parameters can be saved and transferred to different users for the reproduction of detection results in different laboratories. To demonstrate the flexibility of our developed stained colour detection technique, the tool has been extended to the analysis of liver cirrhosis. It is a novel method based on our statistical colour detection model which greatly improves the analysis accuracy and reduces the time cost.

Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation.

KEYWORDS

Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation

Background: The rare nature of male breast cancer (MBC) has led to its management being guided by the extensive research conducted in the field of female breast cancer (FBC). The aim of this study was to evaluate MBC at both protein and molecular level to improve understanding of its pathology. Methodology: Immunohistochemistry analysis was performed in MBC (n=428) TMAs for 18 biomarkers (ERα, ERβ1, ERβ2, ERβ5, Total PR, AR, CK5/6, CK14, CK18, CK19, p53, Bcl-2, Her2, E-cadherin, Ki67, Survivin, Prolactin and FOXA1). The manual scoring of ERα and Ki67 was correlated with a fully automated immunohistochemistry image analysis system (ImmunoRatio™). Finally gene expression profiling (GEP) was undertaken in matched MBC (n=15) and FBC (n=10) samples. Results: There was poor 5 year overall survival (OS) in CK18 and CK19 negative patients (p= 0.05; p= 0.003), as well as poor 10 year OS in CK19 negative patients (p= 0.002). Age (p= 0.001) and nodal status (p= 0.04) was found to be independent predictors of OS at 5 years. There was significant correlations between manual and ImmunoRatio™ ERα (ρ= 0.872; p= 0.000) and Ki67 (r= 0.675; p= 0.000) scores. However due to a low measure of agreement it was not possible to validate Ki67 scoring using ImmunoRatio™. The functional enrichment analysis of GEP data using less stringent criteria (p < 0.05) identified 735 differentially expressed genes. The data analysis showed up-regulation of genes involved in ECM synthesis, degradation and re-modelling in MBC. The end product of one of the up-regulated genes (Fibronectin (FN1)) was validated in the MBC cohort with high fibronectin expression (60%) being positively associated with nodal status and showed a trend towards poor 5 year OS (p= 0.06). Conclusion: In MBC, epithelial cytokeratins, especially CK19 was found to be of prognostic significance. The extracellular matrix remodelling associated genes were found to be up-regulated in MBC. Fibronectin, end product of one of the up-regulated gene was found to have prognostic significance in MBC.

Primary scalp alopecia is classically divided into cicatricial (scarring) and non-cicatricial (non-scarring). Challenging cases are assessed with a scalp biopsy. The use of both horizontal and vertical sections (HoVert sections) has dramatically improved the accuracy of histopathological diagnosis. In this work, we have used immunostaining to address diagnostic difficulties, which persist despite all currently available tools. We performed an immunostain panel (CD3, CD4, CD8 and CD20) in order to distinguish pattern hair loss from alopecia aerate in cases which do not have the usual peribulbar lymphocytic infiltrate and showed that CD3+ T-lymphocytes within the empty fibrous follicular tracts favor a diagnosis of alopecia areata. We performed CD123 in order to distinguish lichen planopilaris from alopecia lupus erythematosus in cases with only a superficial lymphocytic infiltrate and an uninvolved interfollicular epidermis and showed that clusters of CD123+ plasmacytoid dendritic cells favor a diagnosis of lupus erythematosus. We performed cytokeratin 15 in order to assess whether the loss of the follicular bulge stem cells has diagnostic value in cicatricial alopecia and demonstrated that the loss of cytokeratin 15+ bulge stem cells is identified in lichen planopilaris, frontal fibrosing alopecia, and lupus erythematous, so cytokeratin 15 has no diagnostic value. We have attempted to integrate the new concepts and our findings into the traditional classifications of alopecia and proposed a new diagnostic algorithm. In conclusion, immunostaining combined with HoVert grossing advances the accuracy of histopathological diagnosis of primary scalp alopecia.
L’alopécie primitive du cuir chevelu est habituellement classée en cicatricielle et non-cicatricielle. Dans les cas difficiles, la biopsie du cuir chevelu peut aider au diagnostic. L’utilisation de coupes, à la fois verticales et horizontales sur le même spécimen (technique HoVert), a radicalement amélioré le diagnostic histopathologique. Dans ce travail, nous avons utilisé l’immunohistochimie pour évaluer les difficultés diagnostiques qui persistent malgré tous les outils actuels. Nous avons utilisé les CD3, CD4, CD8 et CD20 pour différencier l’alopécie androgénique de la pelade dépourvue de l’infiltrat lymphocytaire péribulbaire habituel et nous avons démontré que la présence de lymphocytes CD3+ dans les travées folliculaires fibreuses est en faveur de la pelade. Nous avons utilisé le CD123 pour différencier le lichen plan pilaire du lupus érythémateux alopécie avec infiltrat lymphocytaire superficiel et sans atteinte de l’épiderme interfolliculaire et nous avons démontré que la présence d’amas de cellules dendritiques plasmacytoïdes CD123+ est en faveur du lupus érythémateux. Nous avons utilisé la cytokératine 15 pour évaluer si la perte des cellules souches du bulge a une valeur diagnostique dans l’alopécie cicatricielle et nous avons démontré que cette perte s’observait de manière identique dans le lichen plan pilaire, l’alopécie frontale fibrosante comme dans le lupus érythémateux et n’avait donc aucune valeur diagnostique. Nous avons tenté d’intégrer les nouveaux concepts et nos données dans les classifications traditionnelles des alopécies et nous avons élaboré un nouvel algorithme diagnostique. L’association des immunomarquages avec la technique HoVert ouvre de nouvelles perspectives dans le diagnostic histopathologique des alopécies primaires du cuir chevelu.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Background: Autoimmune bullous dermatoses present with overlapping clinical features that require histopathological correlation. Immunofluorescence is the most routinely used reliable investigation for diagnosis but requires specialised equipment and is technically sophisticated. Collagen IV immunohistochemistry is reported as a reliable test for the diagnosis of epidermolysis bullosa acquisita whereby It stains the roof of a subepidermal blister and would be expected on the floor in bullous pemphigoid. This technique could be performed as an easily accessible alternative to direct immunofluorescence and has been used anecdotally at our hospital. Aim: To investigate whether collagen IV immunohistochemistry can be used as a reliable histopathological confirmation of bullous pemphigoid. Methods: Two major investigations: 1. A systematic literature search was undertaken of all studies describing the use of collagen IV immunohistochemistry and those comparing it with immunofluorescence in the diagnosis of bullous pemphigoid. 2. A retrospective study of patients diagnosed with bullous pemphigoid over 12 years seen at Groote Schuur Hospital was performed. Patient records that had results for both direct immunofluorescence and collagen IV immunohistochemistry were selected. The positive percentage agreement was calculated. Results: 1. Two studies were found that investigated the use of collagen IV immunohistochemistry in bullous pemphigoid. All reported 33 (100%) cases demonstrated collagen IV at the floor of a subepidermal blister. Of these, 25/25 cases were in agreement with direct immunofluorescence and 7/8 with indirect immunofluorescence which were used as reference standard investigations. 2. In this study, collagen IV was positive in 96% (79/82) of cases and direct immunofluorescence was positive in 85% (72/82) of cases. A positive percentage agreement of 80.5% suggested a strongly positive test accordance. Limitations: 1. The literature search was limited to articles written in english only. 2. The retrospective design and the lack of controls without bullous pemphigoid made it impossible to calculate sensitivity and specificity as well as the kappa statistic. Conclusion: Collagen IV immunohistochemistry is a valid, simple and widely available test which demonstrates accordance with routinely used direct immunofluorescence in the confirmation of bullous pemphigoid. Through clinical and histomorphological correlation, it may be a useful test in resourcelimited settings without facilities for direct immunofluorescence. However, larger controlled studies are warranted to confirm this.

Bioengineering
M.S.
Non-healing wounds, also known as chronic wounds, are defined as wounds that do not show improvement in healing within four weeks. Chronic wounds affect millions of people around the world and health care expenses in the United States can cost more than one billion dollars. Chronic wound healing is a complicated process with different pathologies depending on the patients’ condition. Four highly integrated and overlapping phases compose the wound healing process: hemostasis, inflammation, proliferation, and tissue remodeling. Occurrence of chronic wounds is usually due to unsuccessful progression through the normal stages of healing, and frequently enters a state of pathologic inflammation. Several cell types are involved in the wound healing process. Platelets initiate the coagulation cascade to stop the bleeding. Keratinocytes are able to restore the epidermis after injury. Vascular endothelial cells form the new blood vessels. Neutrophils and macrophages are responsible for phagocytosis and the release of growth factors and cytokines. Fibroblasts secrete collagen to fill the wound gap. Skin or tissue grafting is one of the many ways to treat non-healing wounds. Currently available skin substitutes have been proven successful in clinical trials, but they have room for improvement. Long-term culturing for cellularized scaffolds, risk of transferring disease from allogeneic or xenogeneic sources, and mismatching mechanical properties limit current skin substitutes in clinical applications. Given the disadvantages of those skin substitutes, plant protein can be a potent and attractive replacement material. Plant proteins can be extracted from renewable resources in abundance. Compared to skin substitutes from porcine or bovine sources, plant protein scaffolds do not have issues with immune rejection and can be formed into gels, films, or fibers with good bio-compatibility. Amongst the many plant proteins, soy protein is one of the ideal materials to make skin substitute scaffolds. Soy protein has been confirmed to be bio-active in vivo and in vitro. In this study, soy protein-based tissue scaffolds (SPS) were applied in full thickness excisional wounds in a porcine model. Immunohistochemistry (IHC) analysis was performed for macrophage invasion, newly formed vessel formation, and picrosirius red staining of collagen deposition. Results from IHC analysis show that SPS can accelerate the wound healing process.
Temple University--Theses

Atherosclerotic plaque disruption leading to thrombosis has traditionally been studied as a rupture of a thin fibrous cap over a lipid-laden necrotic core. However, two noteworthy categories of plaques that do not rupture have presented themselves: 1) in mice, plaque rupture is rare if not absent, and 2) in humans, some plaques erode and form a thrombus without rupturing. Current understanding of the biomechanical differences between plaques that rupture and those that do not is incomplete. In this research, we used patient-specific computational biomechanics tools to study differences among these groups. Lesion-specific solid mechanical modeling of murine plaques revealed that the relative distribution of stresses differs considerably between mice and man. In human vulnerable plaques, peak stresses are on the thin fibrous cap over a necrotic core, but in mice the highest stresses are in the media and adventitia, away from the plaque. Whereas atherosclerotic human arteries usually experience neointima formation around the entire circumference of the vessel, mouse plaques tend to be punctate and adjacent lesion-free regions. The difference in mechanical environment suggests that plaque rupture, if possible in mice, is likely not driven by mechanics in the same manner as humans. Similar mechanical modeling of human ruptured and eroded plaques and comparison to histological staining revealed that ruptured plaques exhibit increased levels of inflammatory markers in response to strain in ruptured plaques, but no such response was observed in plaque erosion. This suggests that treatment of inflammation, a current paradigm for care of atherosclerotic patients, may not be an effective approach to mediate plaque erosion. Computational fluid dynamics modeling of patients with plaque erosion revealed no relation between wall shear stress magnitude or direction, further suggesting that the mechanism of plaque erosion differs considerably from that of plaque rupture. Together, these findings suggest that biomechanics can help explain why not all plaques rupture and that different clinical approaches are necessary to address different phenotypes of lesions.

The claustrum is a much neglected nucleus in the brain, whose fuction remains unknown to date. Yet, based on the extensive reciprocal connections it shares with virtually all functional regions of cortex, it most likely serves a far from meaningless purpose. Current hypotheses propose a role in perceptual binding by synchronization of cortical activity. These hypotheses come with a number of assumptions, such as a wide network of connections intrinsic to the claustrum. In this context, gap junctions have been suggested as a means to interconnect portions of the claustrim and to synchronize incoming cortical activity. Neuronal gap junctions have been shown to be involved in supporting synchronous activity and oscillations in areas such as the hippocampus, making them a feasible candidate for such a mechanism in the claustrum. While gap junctions have been described in many areas of the brain, they have not been observed in the claustrum prior to the present study. Set aginst this background, the presence of gap junctions in the claustrum was investigated in this thesis. To this end, an immunohistochemical approach was first used to localize the gap junction protein connexin 36. Once a protocol was established, a small population of connexin 36 expressin neurons was identified in the posterior half of the dorsomedial aspect of the ventral claustrum (also known as the endopiriform nucleus). However, no further labeling was observed throughout the rest of the claustrum. These results were supported by electron microscopy, as putative gap junctions were exclusively observed in an area corresponding to where connexin 36 expression was found. These results represent the first steps in confirming the presenceof the hypothesized gap junction network in the claustrum. Yet, it could be argued that their limited occurrence in both number and location are not entirely in line with current hypotheses predicting a wide gap junction network common to the entire claustrum.

This thesis studies sensory innervation in mammals using an organotypic co-culture of spinal cord-dorsal root ganglion and skeletal muscle of embryonic rat, the histological changes of reinnervated muscle spindles after nerve section and the localisation of the calcium-binding protein calretinin in cat mechanoreceptor organs. The immediate importance of this project concerns the better understanding of how the normal process of development differs from reinnervation following nerve lesion or section. A range of classical and well defined materials and methods as been used in the work described. The thesis Is divided into Ove chapters: Chapter 1 reviews aspects of the mechanosensory organs which have been studied experimentally in relation to their sensory innervation, including proprioceptive muscle spindle development, reinnervation, and finally, the presence of the calcium-binding protein, calretinin in the mechanoreceptor organs. This provides an introduction and background to the work. Chapter 2 describes the organotypic organisation of spinal-cord, dorsal-root ganglia and skeletal muscle co-culture in vitro. Results show that slices of the spinal-cord, dorsal- root ganglia survive well under experimental conditions and can live for several weeks with feeding every 1-3 days. Sensory neurons can develop and grow in a medium without any additional promoting factor. The presence of structurally identifiable synapses indicates that other neurons are also maintained in culture and have functional connections. In the organotypic culture new muscle fibres can form either from the original explant or from the additional explant. In chapter 3 I describe two abnormal endings present in spindles of the tenuissimus of the cat that had been reinnervated following section of the nerve more than one year previously. The reconstruction of the endings of these two spindles supports the hypothesis of modulation of the primary-ending response by the mechanical properties of the intrafusal muscle fibres, rather than by intrinsic properties of the la afferent itself. They further indicate that, in the absence of a la afferent, intrafusal-fibre differentiation can be maintained by a group II afferent. Chapter 4 concerns the localisation of the calcium-binding protein calretinin, which was studied immunohistochemically in the abductor digiti quinti medius muscle of the cat hind limb. The calretinin immunoreactivity was found in some intrafusal fibres, the primary endings and the cqjsule of the muscle spindles and the sensory terminals of tendon organs and Paciniform corpuscles. The present findings contradict a recent hypothesis that calretinin is associated with rapid adaptation, but suggest that calretinin has a specific function in muscle proprioceptors. Finally, Chapter 5 outlines the conclusions of this study and gives some suggestions for continuation of the work in the future.

This work describes 5 groups of tumours seen in routine neuropathological practice in Southampton between 1980 and 1986. The clinical and light microscopic findings in a total of 44 tumours are described. A panel of monoclonal and polyclonal antibodies was used in immunohistochemical studies on each group, and this made important contributions to the diagnosis and characterisation of these tumours. Six primary central nervous system lymphomas were shown to be 5 follicle centre cell lymphomas (Kiel-classification) and 1 T-cell lymphoma. 15 lymphomas causing spinal cord compression were typed as 11 follicle centre cell lymphomas, 3 T-cell lymphomas and 1 lymphoblastic lymphoma. T-cell lymphomas appear to be more likely to be localised in the spine and to have a better prognosis. In all these tumours admixed reactive cell populations were also identified immunohistochemically. Ten primitive neuroectodermal tumours of the cerebrum were examined and immunohistochemistry assisted in their distinction from lymphoma and metastatic carcinoma. It also showed evidence of differentiation in apparently poorly differentiated parts of the tumours. Immunohistochemical studies facilitated the distinction of pleomorphic xanthoastrocytoma from a monstrocellular astrocytoma and a meningeal malignant fibrous histiocytoma. The recognition of this first entity has important prognostic implications. In 10 cerebellar haemangioblastomas a panel of antibodies was used to investigate the histogenesis of the stromal cell component of these tumours. Endothelial and stromal cells were found to be antigenically distinct and neurone specific enolase activity was found in the latter. The implications of this finding are discussed. The studies confirm the important and increasing role played by immunohistochemistry in our understanding of central nervous system neoplasia.

Oesophageal squamous cell carcinoma (OSCC) is a highly malignant tumour that has a poor prognosis and shows marked regional variation in its incidence, implicating environmental factors. South Africa is one of several countries that has areas of high incidence. The exact aetiopathogenesis of OSCC is not well understood. Current environmental risk factors include alcohol, tobacco, human papillomavirus (HPV) infection and nutritional factors including; low intake of Vitamins A, C and riboflavin, lack of fruit and vegetables, ingestion of fungal contaminated foods and consumption of extremely hot beverages. This study was a retrospective immunohistochemical study done on paraffin embedded tissues. The histopathology, grading and staging of all resected squamous cell carcinomas over a twenty one year period from 1982 to 2002, were reviewed. Sixty eight patients were identified; all had an oesophagectomy for OSCC at Groote Schuur Hospital, a tertiary referral centre. Clinical details regarding gender, race, age, smoking or alcohol usage and survival data were collected. Survival data was updated to 23 June 2003. Two paraffin blocks representing OSCC and normal mucosa for each patient were retrieved from the archives in the Division of Anatomical Pathology. In addition, 16 cases of reflux oesophagitis were included for comparison. Initial immunohistochemical staining for HPV (Dako- clone KlH8) was undertaken but the negative results necessitated a shift in the focus of this study to that of cell cycle regulatory proteins. The tissues were evaluated for p53 (Dako - clone D0-7), p2l (Novocastro - clone 4Dl0), cyclin DI (Dako - clone DCS-6) and cyclin E (Novocastro - clone 13A3). Expression was interpreted as positive if 10% or more of the tumour cell population stained. Expression was also stratified into three levels (1, 2 and 3) depending on the percentage positive staining. Normal mucosa did not stain for any of the cell cycle regulators. OSCC stained as follows: 61.8% for p53, 27.9% for p21, 22.1 % for cyclin E and 44.1% for cyclin Dl. Reflux oesophagitis stained as follows: 31.2% for cyclin DI, 12.5% for p21 and 0% for both p53 and cyclin E. Subsequent statistical analysis failed to reveal any prognostic significance to the expression of cell cycle regulators, nor could expression or level of expression be associated with stage, grade, age, gender or alcohol use. There was however a significant relationship between cyclin DI and smoking. In addition, expression of p53 discriminated between malignant and reactive oesophageal lesions. Advancing age proved to be associated with an increased risk of mortality. Lastly, histopathological staging proved to be the most significant prognostic factor in this study.

Immunohistochemistry involves the use of labelled antobodies to detect and visualise the presence of selected antigens. It is widely used in the doagnosis of cancers in which specific antigens are either up-regulated or absent.«br /» We are studying newly-discovered biomarkers such as BAP1, and are suggesting novel uses for them in the diagnosis of cancers such as malignant mesothelioma, cholangiocarcinoma, and gynecological malignancies.«br /» We are also examining the role of microsatellite instability in the diagnosis of colorectal cancer and adrenocortical cancer.

Orientador: Oslei Paes de Almeida
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-11T22:55:27Z (GMT). No. of bitstreams: 1 MartinezMata_Guillermo_D.pdf: 5085284 bytes, checksum: 173edda03dc15aa9efb05b262cd34e77 (MD5) Previous issue date: 2007
Resumo: A região bucomaxilofacial é freqüentemente afetada por lesões císticas e tumorais que apresentam características clínicas e histológicas heterogêneas cuja origem está associada a diversos estágios da odontogênese. Entre as lesões císticas mais freqüentes, temos o queratocisto odontogênico (QO), enquanto o cisto odontogênico ortoqueratinizado (COO) é uma lesão rara. O QO apresenta índices de prevalência e recorrência elevados, sendo a terceira lesão cística mais comum na região orofacial, depois dos cistos radicular e dentígero, podendo estar associada à síndrome de Gorlin (QOSG). O fibroma odontogênico central (FOC) é um tumor odontogênico pouco freqüente, com 72 casos relatados na literatura, que afeta mais comumente a região mandibular de mulheres na terceira década da vida. O objetivo deste trabalho foi analisar as características clínicas, radiográficas, histopatológicas e perfil de marcadores de imunohistoquímica em 66 casos de QO solitários, 26 casos de QOSG, oito casos de COO e 10 casos de FOC. As lesões císticas odontogênicas afetaram em 51% dos casos homens e 49% mulheres. A marcação imunohistoquímica do epitélio mostrou positividade para Ck AE1/AE3, Ck 5, Ck 10, Ck 14 e Ck 19 com diferentes intensidades. A expressão de Bcl-2 foi estatísticamente significante maior para QOSG e QO quando comparados com COO. FOC apresentou-se mais comumente no gênero masculino, correspondendo a 70% dos casos, e afetou predominantemente as regiões posteriores da mandíbula (60%). Dois casos de FOC apresentaram-se como lesões híbridas, associados à lesão central de células gigantes (FOC/LCG). Em todos os casos de FOC o epitélio foi imunoreativo para Ck AE1/AE3, Ck 5, Ck 14 e Ck 19. O estroma de FOC foi positivo apenas para vimentina. As citoqueratinas 1, 7, 8 e 18 assim como HHF35, desmina e proteína S-100 foram negativos. A expressão de Bcl-2 nos FOC foi baixa, menor que 1%. Em conclusão QO, QOSG e COO expressaram perfil semelhante de Ck, mas com padrão distinto para Ck5 e Ck14. A expressão de Bcl-2 foi significantemente mais intensa nos QO e QOSG em relação ao COO. O perfil de expressão nas ilhas epiteliais do FOC para citoqueratinas foram semelhante aos QO, mas a expressão de Bcl-2 foi nula nos casos de FOC, o que pode estar relacionado com a menor taxa de recidiva destas lesões odontogênicas.
Abstract: Jaw bones are frequently affected by tumoral and cystic lesions which present heterogeneus clinical and pathological features associated with diverse stages of odontogenesis. These lesions include odontogenic keratocyst (OK) and rarely odontogenic orthokeratinized cyst (OOC). OK presents high recurrence and prevalence index, being the third lesion affecting the jaw bones, preceded only for radicular and dentigerous cyst; could be associated to Gorlin syndrome. Central odontogenic fibroma (COF) is a rare odontogenic tumor, with only 72 cases reported in the English literature, and is more prevalent in the posterior mandibular region of females in the third decade of life. The purpose of this work was to analyze the clinical, radiographic, histologic and immunohistochemistry profile of 66 cases of OK, 26 cases of OKGS, 26 cases of OOC and 10 cases of COF. Cystic lesions were more prevalent in males (51%) than in females (49%). Epithelial tissue of OK was reactive for Ck AE1/AE3, Ck5, Ck10, Ck14 and Ck19. The Bcl-2 and p53 expression was statistically significant for OK and OKGS when compared with OCC. COF was more common in males (70%) than in females (30%), and in the posterior mandibular regions (60%). Two cases were classified as "hybrid lesions" (COF and CGL). Epithelium of COF was reactive for Ck AE1/AE3, Ck5, Ck14 and Ck19. Stromal cells of FOC were positive for vimentin and negative for HHF-35, desmin and S-100 protein. Bcl-2 expression was negative. In conclusion, OK, OKGS and OOC have a similar immunohistochemical profile, although Ck5 and Ck14 present different pattern of expression among OK, OKGS and OOC. Bcl-2 index for OK and OKGS was higher than that of OCC. Epithelial nests of COF were positives for Ck's similarly to OK, OKGS and OOC. The index expression of Bcl-2 in COF was low when compared with that of cystic lesions, and could be related with the low index of recurrence of this tumor.
Doutorado
Patologia
Doutor em Estomatopatologia

Orientador: Julieta Rodini Engracia de Moraes
Banca: Ana Maria Ferreira Roselino
Banca: Angela Cleusa de Fatima Banzatto de Carvalho
Banca: Carlos Noriyuki Kaneto
Banca: Gilson Pereira de Oliveira
Resumo: A leishmaniose é uma enfermidade provocada por protozoários do gênero Leishmania, que pode produzir manifestações cutâneas, mucocutâneas ou viscerais. Os objetivos deste ensaio foram os de identificar a espécie Leishmania (Leishmania) chagasi, utilizando o método PCR-RFLP em amostras de tecido de cães com leishmaniose visceral; seqüenciar o fragmento amplificado de DNA das amostras de linfonodos de diferentes animais, em busca de homologia e polimorfismos; comparar os métodos de imunoistoquímica e de PCR dos rins, na identificação da Leishmania e comparar as técnicas sorológicas em relação à PCR como auxílio diagnóstico da leishmaniose. Para tanto, foram utilizadas amostras de 48 cães com leishmaniose, diagnosticados por meio de testes IFI, ELISA e por PCR. Realizou-se PCR com amostras de linfonodo poplíteo, baço e rins, utilizando 'primers' específicos para o gênero Leishmania. A partir de amostras amplificadas, o DNA foi submetido à digestão enzimática (técnica PCR-RFLP) com as enzimas Hae III, Bsr I e Rsa I. Para o seqüenciamento de DNA, produtos de PCR foram amplificados com 'primer sense', precipitados e submetidos ao seqüenciamento automático. Com fragmentos histológicos renais, realizou-se a técnica imunoistoquímica utilizando anticorpo policlonal anti-Leishmania produzido em coelho. O método PCR-RFLP permitiu identificar a espécie Leishmania (Leishmania) chagasi em todas as amostras de DNA dos diferentes tecidos. No seqüenciamento de DNA, os fragmentos amplificados mostraram-se pertencentes às espécies causadoras de leishmaniose visceral, porém não foi possível diferenciá-las. A técnica de imunoistoquímica mostrou presença de formas amastigotas íntegras em meio ao infiltrado inflamatório intersticial em dois (4%) dos 48 animais avaliados, enquanto a PCR confirmou Leishmania sp em 77% dos cães. Conclui-se que a técnica... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Leishmaniasis is a disease caused by organisms belonging to the genus Leishmania. Clinical forms of leishmaniasis are found as being cutaneous, mucocutaneous or visceral. The objectives of the present study were to identify the Leishmania (Leishmania) chagasi species in tissue specimen from dogs presenting visceral disease diagnosed by PCR-RFLP assay; to determine the fragment sequence (120bp) from lymph node samples from different animals searching for homologies and polymorphism; and to compare immunohistochemistry method to PCR assay with renal tissue and Leishmania local identification. Forty eight samples from dogs clinically diagnosed positive to leishmaniasis by IFAT, ELISA and PCR assays were used in this study. The PCR were performed with samples of popliteo lymph node, spleen and kidneys, and specific oligonucleotides to genus Leishmania. PCR products were digested with enzymes Hae III, Bsr I and Rsa I. The nucleotide sequences were determined automatically. For immunohistochemical purposes the sections of kidneys and anti-Leishmania polyclonal antibody were used. PCR-RFLP assay allowed us to identify the Leishmania (Leishmania) chagasi specie in all DNA samples from different tissues samples. DNA sequencing revealed products amplified belonging to specie responsible to cause visceral leishmaniasis, but it was not possible to distinguish the species. The immunohistochemical study revealed the presence of amastigotes organisms on intersticial inflammatory infiltrate from two dogs (4%), while the PCR assay detected the Leishmania sp in 37 dogs (77%). Based on the PCR-RFLP results, we concluded that it characterized as being Leishmania (Leishmania) chagasi species, etiologic agents of visceral leishmaniasis, in this group of animals; DNA sequence presented homology of 55 bp among all Leishmania spp studied. Additionally, the PCR performed... (Complete abstract click electronic access below)
Doutor

The incidence of cutaneous malignant melanoma has increased dramatically in Caucasians the last few decades, an increase that is partly explained by altered sun exposure habits. For the individual patient, with a localized disease, the tumor thickness of the excised lesion is the most important prognostic factor. However, there is a need to identify characteristics that can place patients in certain risk groups. In this study, the protein expression of multiple proteins in malignant melanoma tumors was studied, with the aim of identifying potential new candidate biomarkers. Representative samples from melanoma tissues were assembled in a tissue microarray format and protein expression was detected using immunohistochemistry. Multiple cohorts were used and for a subset of proteins the expression was also analyzed in melanocytes in normal skin and in benign nevi. The immunohistochemical staining was evaluated manually and for part of the proteins also with an automated algorithm. The protein expression of STX7 was described for the first time in tumors of the melanocytic lineage. Stronger expression of STX7 and SOX10 was seen in superficial spreading melanomas compared with nodular malignant melanomas. An inverse relationship between STX7 expression and T-stage was seen and between SOX10 expression and T-stage and Ki-67, respectively. In a population-based cohort the expression of MITF was analyzed and found to be associated with prognosis. Twenty-one potential biomarkers were analyzed using bioinformatics tools and a protein signature was identified which had a prognostic value independent of T-stage. The protein driving this signature was RBM3, a protein not previously described in malignant melanoma. Other markers included in the signature were MITF, SOX10 and Ki-67. In conclusion, the protein expression of numerous potential biomarkers was extensively studied and a new prognostic protein panel was identified which can be of value for risk stratification.

Background Prostate cancer (PCa) is the most frequently diagnosed malignant disease among adult males in the USA and the second leading cause of cancer deaths in men. Due to the lack of diagnostic tools that are able to differentiate highly malignant and aggressive cases from indolent tumors, overtreatment has become very common in the era of prostate specific antigen (PSA) screening. New diagnostic methods to determine biological status, malignancy, aggressiveness and extent of PCa are urgently needed. 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HRMAS MRS) can be used to establish PCa metabolomic profiles while preserving tissue architecture for subsequent histopathological analysis. Immunohistochemistry (IHC), as opposed to conventional histopathology methods, has the potential to provide objective, more accurate and quantitative knowledge of tissue pathology. This diagnostic- accuracy study sought to evaluate a novel approach to quantitatively identify metabolomic markers of PCa by exploring the potential of PCa immunomarkers to quantify metabolomic profiles established by 1H HRMAS MRS. Material and Methods 1H HRMAS MRS was performed on tissue samples of 51 prostate cancer patients using a 14.1 Tesla NMR spectrometer (BRUKER Biospin, Billerica, MA) with a rotor synchronized CPMG pulse sequence. Spectral intensities of 36 regions of interest were measured as integrals of curve fittings with Lorentzian-Gaussian line shapes. Immunohistochemistry (IHC) was carried out following the spectroscopy scan, using three prostate immunomarkers to identify cancerous and benign glands: P504S (Alpha-methylacyl-CoA-racemace), CK903 (high-molecular weight cytokeratin) and p63. The immunostaining quality following 1H HRMAS MRS was evaluated and compared to unscanned sections of the same sample, to verify the stability and accessibility of the proposed immunomarkers. IHC images were automatically and quantitatively evaluated, using a quantitative image analysis program (QIAP), to determine the percentage of cancerous and benign epithelia in the tissue cross- sections. The results of the program were validated by a correlation with the results of a quantitative IHC review and quantitative conventional histopathology analysis performed by an experienced pathologist. Ultimately, spectral intensities and the cancer epithelium percentage, obtained from quantitative immunohistochemistry, were correlated in order to validate PCa metabolomic markers identified by 1H HRMAS MRS. Patient outcomes and incidence of recurrence were determined by retrospective review of medical records five years after initial surgery. Categories of recurrence were correlated to spectral intensities to explore potential metabolomic markers of recurrence in the cohort. Results Immunostainings with P504S and CK903 showed excellent staining quality and accessibility following 1H HRMAS MRS, suggesting these markers to be suitable for the presented quantitative approach to determine metabolomics profiles of PCa. In contrast, the quality of p63 IHC was impaired after previously performed spectroscopy. IHC using the immunomarkers P504S and CK903 on adjacent slides was found to present a feasible quantitative diagnostic method to distinguish between benign and cancerous conditions in prostate tissue. The cancer epithelium percentage as determined by QIAP showed a significant correlation to the results of quantitative IHC analysis performed by a pathologist (p < 0.001), as well as to a quantitative conventional histopathology review (p = 0.001). The same was true for the benign epithelium percentage (p < 0.001 and p = 0.0183), validating the presented approach. Two metabolomic regions showed a significant correlation between relative spectral intensities and the cancer epithelium percentage as determined by QIAP: 3.22 ppm (p = 0.015) and 2.68 ppm (p = 0.0144). The metabolites corresponding to these regions, phosphocholine and citrate, could be identified as metabolomic markers of PCa in the present cohort. 45 patients were followed for more than 12 months. Of these, 97.8% were still alive five years after initial surgery. 11 patients (24.4%) experienced a recurrence during the follow- up time. The categories of recurrence showed a correlation to the spectral intensities of two regions, 2.33 – 2.3 ppm (p = 0.0403) and 1.28 ppm (p = 0.0144), corresponding to the metabolites phosphocreatine and lipids. Conclusion This study introduces a method that allows an observer-independent, quantitative analysis of IHC to help establish metabolomic profiles and identify metabolomic markers of PCa from spectral intensities obtained with 1H HRMAS NMR Spectroscopy. The immunomarkers P504S and CK903 have been found suitable IHC analysis following 1H HRMAS MRS. A prospective in vivo application of PCa metabolite profiles and metabolomic markers determined by the presented method could serve as highly sensitive, non- invasive diagnostic tool. This observer- independent, computer- automated, quantitative analysis could help to distinguish highly aggressive tumors from low-malignant conditions, avoid overtreatment and reduce risks and complications for cancer patients in the future. Further studies are needed to verify the identified PCa metabolomic markers and to establish clinical applicability
Einführung Prostatakrebs ist eine häufigsten Krebserkrankungen in den USA und die zweithäufigste malignom- assoziierte Todesursache männlicher Patienten weltweit. Seit der Einführung des Prostata- spezifischen Antigen (PSA)- Screeningtests wird diese Krebsart in früheren Stadien diagnostiziert und therapiert, wodurch die Mortalitätsrate in den letzten Jahren deutlich reduziert werden konnte. Da moderne diagnostische Methoden bislang jedoch nicht ausreichend in der Lage sind, suffizient zwischen hochmalignen und weniger aggressiven Varianten dieses bösartigen Krebsleidens zu unterscheiden, werden häufig auch Patienten aggressiv therapiert, deren niedriggradiges Prostatakarzinom keine klinische Relevanz gehabt hätte. Es besteht daher ein großes wissenschaftliches Interesse an der Entwicklung neuer diagnostischer Methoden zur akkuraten Bestimmung von biologischem Status, Malignität, Aggressivität und Ausmaß einer Prostatakrebserkrankung. \\\\\\\"1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy\\\\\\\" (1H HRMAS MRS) ist eine vielversprechende diagnostische Methode, welche es ermöglicht, metabolomische Profile von Prostatakrebs zu erstellen, ohne die Gewebsstruktur der analysierten Proben zu zerstören. Durch anschließende histopathologische Begutachtung lassen sich die erstellten Metabolitprofile validieren und evaluieren. Im Gegensatz zu konventionellen histopathologischen Methoden können durch immunhistochemische Verfahren dabei objektivere, akkuratere und quantifizierbare histopathologische Erkenntnisse gewonnen werden. Die vorliegende Studie präsentiert einen neuentwickelten diagnostischen Ansatz zur quantitativen Bestimmung von metabolomischen Markern von Prostatakrebs, basierend auf der Durchführung von 1H HRMAS NMR Spektroskopie und quantitativer Immunhistochemie. Material und Methoden Einundfünfzig Gewebsproben von Prostatakrebspatienten wurden mittels 1H HRMAS MRS an einem 14.1 T BRUKER NMR Spektrometer unter Einsatz einer CPMG-Pulssequenz untersucht. Spektrale Intensitäten in 36 Metabolitregionen wurden gemessen. Anschließend wurden die analysierten Gewebeproben mit drei Immunfärbemarkern für sowohl malignes (P504S, Alpha-methylacyl-CoA-racemase) als auch benignes (CK903, High-molecular weight cytokeratin, und p63) Prostatagewebe angefärbt und quantitativ mit Hilfe eines Bildanalyseprogramms (QIAP) ausgewertet. Die Anwendbarkeit und Auswertbarkeit der genannten Immunomarker nach Spektroskopie wurde evaluiert und mit der Färbungsqualität von nicht- gescannten Schnitten verglichen. Die Resultate der automatischen Auswertung durch QIAP konnten durch einen erfahrenen Pathologen in einer quantitativen Analyse der Immunfärbungen sowie konventioneller histologischer Färbungen derselben Gewebsproben validiert werden. Die spektralen Intensitäten aus den Messungen mit 1H HRMAS MRS wurden mit den korrespondierenden Ergebnissen der quantitativen Auswertung der Immunfärbungen korreliert, um metabolomische Marker von Prostatakrebs zu identifizieren. Der klinische Verlauf und die Rezidivrate der Patienten wurden 5 Jahre nach der initialen Prostatektomie retrospektiv bestimmt. Rezidivkategorien wurden erstellt und mit den bestimmten spektralen Intensitäten korreliert, um metabolomische Marker für das Auftreten von Prostatakrebsrezidiven zu identifizieren. Ergebnisse Die Immunfärbungen mit P504S und CK903 zeigten exzellente Qualität und Auswertbarkeit nach vorheriger 1H HRMAS MRS. Beide Marker eigneten sich zur Durchführung von quantitativer Immunhistochemie an spektroskopierten Gewebeproben. Im Gegensatz dazu war die Qualität der Immunfärbungen mit p63 nach Spektroskopie vermindert. Quantitative Immunfärbungen unter Einsatz der Immunmarker P504S und CK903 stellten eine praktikable diagnostische Methode dar, um zwischen malignen und benignem Prostatagewebe zu unterscheiden. Der Anteil von bösartig verändertem Prostatagewebe, bestimmt durch QIAP, korrelierte signifikant mit den Ergebnissen der quantitativen Analyse der Immunfärbungen durch den Pathologen (p < 0.001), sowie mit der quantitativen Auswertung der konventionellen histopathologischen Färbung (p = 0.001). Ebenso ließ sich die Bestimmung des Anteils von benignem Gewebe mit QIAP zu den Ergebnissen der pathologischen Analyse korrelieren (p < 0.001 und p = 0.0183). Für zwei metabolomische Regionen konnte ein signifikante Korrelation zwischen relativen spektralen Intensitäten, bestimmt mit 1H HRMAS NMR Spektroskopie, und dem Anteil von malignem Epithelium in derselben Gewebeprobe, ermittelt durch QIAP, festgestellt werden: 3.22 ppm (p = 0.015) und 2.68 ppm (p = 0.0144). Die zu diesen Regionen korrespondierenden Metaboliten, Phosphocholin und Zitrat, konnten als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Die retrospektiven Analyse der klinischen Daten der Patienten fünf Jahre nach Prostatektomie ergab eine Überlebensrate von 97.8%. Elf dieser Patienten (24.4%) erlitten ein Rezidiv ihrer Erkrankung. Die bestimmten Rezidivkategorien korrelierten signifikant mit zwei metabolomischen Regionen (2.33 – 2.3 ppm, p = 0.0403 und 1.28 ppm, p = 0.0144), welche zu den Metaboliten Phosphokreatin und Lipiden korrespondierten. Schlussfolgerung Die vorliegende Studie präsentiert einen diagnostischen Ansatz zur objektiven und quantitativen Bestimmung metabolomischer Marker von Prostatakrebs unter Verwendung von 1H HRMAS MRS und Immunhistochemie. P504S und CK903 eignen sich als Immunmarker für quantitative Immunfärbungen nach vorheriger Durchführung von 1H HRMAS MRS. Die Metaboliten Phosphocholin und Zitrat konnten in der vorliegenden Patientenkohorte als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Eine mögliche in vivo Anwendung der gefundenen metabolomischen Marker könnte als hochsensitives, objektives und nicht- invasives diagnostisches Werkzeug der Prostatakrebsdiagnostik dienen. Der vorliegende untersucherunabhängige, automatisierte und quantitative diagnostischer Ansatz hat das Potential, zwischen hochmalignen und weniger aggressiven Krebsfällen zu unterscheiden und somit unnötige Risiken und Komplikationen für Prostatakrebspatienten zu reduzieren. Weitere Untersuchungen sind notwendig, um die identifizierten metabolomischen Marker zu verifizieren und eine klinische Anwendung zu etablieren

Corneal transplantation carries a high rate of success in the domestic cat and is an indicated treatment for specific corneal diseases in this species. The potential for iatrogenic transmission of viral diseases is a well-recognized problem in human corneal transplantation programs and screening donors for certain diseases is routine. Feline leukemia virus (FeLV) is a common agent of disease in domestic cats and available blood tests are highly effective in identification of infected individuals. This study investigates the presence of FeLV within corneal tissues of FeLV infected cats. Seventeen cats were identified to be positive for serum p27 antigen by enzyme-linked immunosorbent assay (ELISA). Twelve of these individuals were found to be positive on peripheral blood by immunofluorescent antibody (IFA) testing. Seventeen ELISA negative cats were identified to serve as negative controls. Full thickness corneal specimens were collected from all subjects and analyzed for the presence of FeLV proviral DNA and gp70 antigen by polymerase chain reaction (PCR) and immunohistochemical (IHC) testing, respectively. Eleven (64.7%) positive corneal PCR results were obtained from 17 ELISA positive cats. Of 12 cats which were both ELISA and IFA positive on peripheral blood, 10 (83.3%) had positive corneal PCR results. All corneal tissues from ELISA negative subjects were PCR negative. IHC staining of corneal sections revealed the presence of FeLV gp70 in corneal tissues of nine (52.9%) ELISA positive cats. Of the 12 cats which were both ELISA and IFA positive on peripheral blood, 8 (66.7%) had positive corneal IHC results. Positive IHC staining was localized to the corneal epithelium. Corneal tissues of all ELISA negative cats and all IFA negative cats were negative on IHC testing. This study reveals FeLV to be present within the corneal epithelium of some FeLV infected cats. Screening potential corneal donors for this virus is warranted. This work was funded by grants from the American College of Veterinary Ophthalmologists, the Virginia Veterinary Medical Association Pet Memorial Fund, and the DSACS Quick Response Fund.
Master of Science

Medulloblastoma is the most frequently occurring childhood malignant brain tumour, affecting 1 of 5 children presenting with a brain tumour, between the ages of 0 and 9 years. The basic prognostic stratification that relies on clinical and histological findings alone has proven unsatisfactory as an outcome predictor. Distinct molecular genetic profiles have been described, with four molecular variants of medulloblastoma with specific demographic and prognostic features. These are the WNT subgroup, SHH subgroup, Group 3 and Group 4 tumours. The aim of this study was to describe the expression status of β-catenin, and MYCN, using IHC and FISH respectively, and to correlate these findings with clinico-pathological and demographic characteristics and clinical outcome. Materials and Methods This study was a nested retrospective analytical study, reviewing 54 cases of childhood medulloblastoma diagnosed between 1988 and 2014. Results Classic histology accounted for 40.7% of cases, LCA 37%, ND 16.7% and 5.6% MBEN). Based on β-catenin IHC, the WNT subgroup accounted for 16.7% of cases. This group had no mortalities or recurrences. Seven patients showed amplification of MYCN gene. The SHH group, defined by ND/MBEN histology and/or MYCN amplification, accounted for 27.7% of patients. Non-WNT/non-SHH tumours 30 patients (55.6%) showed a male predilection, and accounted for 37.5% recurrences and 50%. mortalities also falling in this group. Conclusions Nuclear β-catenin identifies WNT tumours. Nodular desmoplastic morphology is useful in identifying some, but not all cases of SHH group medulloblastomas. MYCN positive tumours also showed classical, and LCA morphology.. Patients of all the beta-catenin positive cases were free of recurrence and alive at last follow up. Patients with MYCN amplification and non-ND histology (LC/A or classic) had poorer outcomes than patients with ND histology. One patient showed both MYCN amplification and nuclear β-catenin translocation, and had good clinical outcome. This finding requires validation with other molecular techniques.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Fundação para o Desenvolvimento da UNESP (FUNDUNESP)
O objetivo deste trabalho foi o de avaliar a cicatrização óssea de fraturas experimentais do radio de cães, tratadas ou não com o PRP autógeno, por meio de estudos radiográfico, densitométrico e histológico; bem como avaliar a expressão dos fatores de crescimento do PRP. Foram utilizados 21 cães inicialmente agrupados de acordo com o tempo de colheita de biopsia: aos sete dias (n=10) ou 60 dias (n=11) que foram alocados aleatoriamente em dois grupos experimentais: o grupo controle (G-controle, n=11) e o grupo PRP (G-PRP, n=10). Todos os animais foram submetidos à osteotomia e osteossíntese (fixador esquelético externo) do rádio direito, gerando-se um “gap” de 2,0mm, que foi preenchido ou não com PRP. Os estudos radiográficos e densitométricos foram realizados no pós-operatório imediato e até 60 dias de pós-operatório. As avaliações histológicas e imunoistoquímicas foram realizadas aos sete e 60 dias. Os dados encontrados foram tratados estatisticamente (p<0,05). Houve diferença significativa nas avaliações radiográficas e densitométricas entre os grupos. A avaliação histológica evidenciou uma cicatrização óssea mais avançada aos 60 dias no G-PRP e união óssea tardia no G-controle. Houve imunomarcação intensa do PDGF-B e TGF-β no G-PRP aos sete e 60 dias de pós-operatório. Conclui-se, que o PRP pode ser utilizado como terapia adjuvante, pois promoveu melhor cicatrização óssea em fraturas experimentais (“gap” de 2,0mm) do radio de cães tratadas com fixador esquelético externo. Ainda houve maior expressão do PDGF-B e TGF-β nos períodos, precoce e tardio, dos animais tratados com PRP
The present article aimed to assess bone healing of experimental radial fractures, treated or not with autologous PRP, by means of radiographic, densitometric and histological studies and evaluate the expression of growth factors in PRP. Were used 21 dogs initially grouped according to the time of biopsy collection: seven days (n = 10) or 60 days (n = 11) were randomly assigned to two groups: the control group (G-control, n = 11) and the PRP group (G-PRP, n = 10). All animals underwent osteotomy and fixation (external skeletal fixation) of the right radius, generating a gap of 2.0 mm, which was filled or not with PRP. Radiographic and densitometry studies were performed in the immediate postoperative period and to 60 days after the surgery. The histological and immunohistochemical evaluations were performed at seven and 60 days. The data were treated statistically (p <0.05). There were significant differences in densitometric and radiographic evaluations between the groups. Histological evaluation showed a more advanced bone healing at 60 days in G-PRP and bone union late in the G-control. There was intense expression of PDGF-B and TGF-β in G-PRP from seven to 60 days postoperatively. It is concluded that the PRP can be used as adjuvant therapy, because it provided better bone healing in experimental fractures (gap of 2.0 mm) radius of dogs treated with external skeletal fixation. Although there was a higher expression of PDGF-B and TGF-β in periods, early and late, the animals treated with PRP

Kidney transplants from both living-related (LRD) and living unrelated (LURD) donors have superior function and survival than transplants from cadaver donors. This may be unsurprising as kidneys from living donors are procured under optimal conditions, from healthy donors with minimal ischaemia times. In contrast, cadaver kidneys are obtained from traumatised donors and may experience extended periods of cold ischaemic storage before transplantation. An immunohistochemical analysis has been performed on biopsies obtained before, and immediately after transplantation, to investigate the potential causes of early inflammatory events associated with cadaver renal transplantation that may influence subsequent graft outcome. An immunohistochemical analysis of biopsies obtained before transplantation demonstrated upregulated expression of endothelial E-selectin and proximal tubular expression of ICAM-1, VCAM-1 and HLA Class II antigens in cadaver donor kidneys. Analysis of donor parameters demonstrated that traumatic physiological events experienced in intensive care around the time of brain death were significantly associated with the induction of proinflammatory antigens. Antigen induction in cadaver donor kidneys before transplantation was significantly associated with early acute rejection. Furthermore, in cadaveric kidneys with long cold ischaemia times, glomerular neutrophil infiltration and deposition of activated platelets expressing P-selectin on intertubular capillaries were detected following reperfusion, in association with impaired short and long term graft function. Expression of inflammatory mediators were absent in all LRD renal allografts before and after reperfusion. A clinical trial was performed to determine whether ischaemia/reperfusion injury may be ameliorated by reflushing cadaver kidneys after cold storage to remove harmful products that may have accumulated in the vessel lumen. Reflushing did not prevent the inflammatory events observed after reperfusion or improve graft function. Therefore, a novel, oxygen free radical scavenger (lec-SOD) was obtained to assess its potential efficacy in preventing ischaemia/reperfusion injury. Lec-SOD bound with high affinity to macro- and microvascular endothelial cells under cold hypoxic conditions following incorporation into Marshall's preservation solution, significantly inhibiting cold hypoxia induced cell death, adhesion molecule induction and neutrophil adhesion. Furthermore, preservation of kidneys with lec- SOD for 18 hr in an experimental model of chronic renal allograft rejection, significantly attenuated neutrophil infiltration and MHC Class I induction day 1 post-transplant, with improved long term renal function. The results presented in this Thesis demonstrate that donor factors and cold ischaemia/ reperfusion injury elicit an early inflammatory response that may influence graft outcome of cadaver kidneys. Refinements in donor management and organ preservation may limit the deleterious effects of ischaemia/reperfusion injury in cadaver renal allografts, increasing graft survival to that observed in living donor transplantation.

An anti-G"11 antibody was used to label neuronal cilia throughout the rat central nervous system. Immunoreactive cilia were observed in every examined region of the rat CNS, but not in monkey or mouse tissue. Antibodies to G"q and G"q/11 failed to label cilia. Immunoreactive cilia were observed as early as postnatal day 0 in spinal tissue, and postnatal day 3 in hypothalamic tissue. There was a statistically significant negative correlation between a region's mean cilium length and that region's distance to the nearest ventricle; regions nearest ventricles were those with the longest cilia. This correlation suggests neuronal cilia may function as chemosensors, detecting substances as they move out from the cerebrospinal fluid and into the extracellular space of the brain.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2010.
Committee Chair: Griffin, Paul; Committee Member: Butera, Robert; Committee Member: Halpern, Michael; Committee Member: Nichols, Richard; Committee Member: Vidakovic, Brani. Part of the SMARTech Electronic Thesis and Dissertation Collection.