O linfonodo é um órgão linfoide secundário que apresenta uma arquitetura altamente organizada com diferentes compartimentos para tipos celulares específicos. Dentre as células estruturais que compõem este órgão, as células estromais como células fibroblásticas reticulares (FRCs) e células duplo negativas (DNCs) parecem ter papel importante na modulação da resposta imunológica e na tolerância periférica. As FRCs são caracterizadas pela expressão de podoplanina (gp38, PDPN) e localizam-se principalmente na zona de células T, enquanto as DNCs (gp38-) apresentam fenótipo, localização e função pouco descritos. Embora estas células tenham sido muito estudadas em modelos murinos os estudos sobre FRCs e DNCs humanas são escassos e, portanto nosso estudo deve contribuir para a compreensão da biologia e a função dessas células, podendo favorecer o conhecimento sobre a eficiência e as disfunções da resposta imune no linfonodo. Com esse intuito, isolamos e caracterizamos fenotípica e funcionalmente as FRCs e DNCs de linfonodos de pacientes com câncer, diverticulite e doadores de fígado. Nossos resultados mostraram a integridade e a distribuição celular no linfonodo. As células aderentes derivadas dos linfonodos estudados preecheram todos os critérios internacionais de caracterização de estroma, e, portanto, foram consideradas células estromais. Através da expressão de gp38 identificamos duas subpopulações de celulas estromais: FRCs (gp38+ e CD31-) e DNCs (gp38- e CD31-) e verificamos que as frequências destas células variam entre as amostras, sugerindo que a doença pode interferir na composição celular estromal dos linfonodos. As duas populações celulares foram estimuladas com citocinas inflamatórias como IFN-y ou TNF-alfa + IL-1beta por 24 e 48 horas e avaliadas quanto à expressão gênica e proteica. Em condições homeostáticas, genes relacionados com a indução e controle da proliferação foram diferencialmente expressos nas FRCs e DNCs, este dado foi confirmado in vitro, uma vez que as FRCs apresentaram maior potencial proliferativo em relação às DNCs. O estímulo com IFN-y induziu aumento de expressão nas DNCs e FRCs para citocinas, quimiocinas, moléculas de histocompatibilidade e moléculas envolvidas na regulação da resposta imunológica. Em resposta ao estímulo com TNF-alfa +IL-1beta, observamos aumento na expressão de moléculas comuns ao estímulo com IFN-?, entretanto, também observamos expressão de moléculas de citocinas, quimiocinas inflamatórias e moléculas de histocmpatibilidade especificamente relacionados a este sinal em ambas as populações. Em conjunto, nossos dados sugerem que DNCs e FRCs apresentam diferenças no perfil de resposta segundo os estímulos inflamatórios aos quais estão expostas, aumentando a expressão diferencial de moléculas envolvidas na regulação positiva e negativa da resposta imune / The lymph node is a secondary lymphoid organ that has a highly organized architecture with different compartments for specific cell types. Among the structural cells that comprise this organ, stromal fibroblastic reticular cells (FRCs) and double negative cells (DNCs) seems to play an important role in modulating the immune response and peripheral tolerance. FRCs are characterized by podoplanin (gp38, PDPN) expression and are located mainly in the T cell zone, while DNCs (gp38-) present phenotype, location and function not well described. Although these cells have been studied in murine models, studies on human FRCs and DNCs are limited and therefore our study should contribute to the understanding of biology and function of these cells and should promote knowledge of efficiency and disorders in the lymph node immune response. For this purpose, we have isolated and characterized phenotypic and functionally lymph nodes derived FRCs and DNCs from patients with cancer, diverticulitis and liver donors. Our results showed lymph node integrity and its cellular distribution. Adherent cells lymph nodes-derived fullfill the international criteria for stroma characterization, and therefore, they have been considered stromal cells. Using gp38 expression we were able to identify two stromal cells subpopulations: FRCs (gp38 + and CD31-) and DNCs (gp38- and CD31-) and found that this cells frequency varies among samples, suggesting that the disease may interfere with lymph nodes stromal cell composition. These two cells populations were stimulated with inflammatory cytokines such as IFN-y or TNF-alfa + IL-1beta for 24 and 48 hours and evaluated for gene and protein expression. In homeostatic conditions, genes involved in the induction and control of proliferation were differentially expressed by FRCs and DNCs, this data has been confirmed in vitro, since the FRCs showed higher proliferative potential compared to DNCs. IFN-y stimulation induced increase DNCs and FRCs expression for cytokines, chemokines, histocompatibility molecules and molecules involved in regulating the immune response.In response to TNF-alfa + IL-1beta stimulation, we observed common molecules expressed by the IFN-? stimulation, however, we also observed expression of cytokines, chemokines and histocompatibility molecules specifically related to this signal in both cells populations. Together, our data suggest that DNCs and FRCs differ in the response profile according to inflammatory stimuli to which they are exposed, increasing the differential expression of molecules involved in the positive and negative regulation of immune response

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

This Ph.D. dissertation examined the effects of bacterial DNA on immune surveillance in ovine lymph nodes (LN). The first study identified long-term changes in lymph nodes following exposure to plasmid DNA. Treated lymph nodes were heavier and had larger medullary areas. Furthermore, medullary cord thickness and medullary sinus width as well as germinal center size and number were increased in plasmid treated lymph nodes.<p>To determine whether bacterial DNA altered cell trafficking through lymph nodes, the efferent lymphatic of the prescapular LN of sheep was cannulated and cells were collected. Intradermal injection of as little as 4 ìg of plasmid DNA expressing the green fluorescent protein of jellyfish (eGFP) caused a marked increase in the cell trafficking through the prescapular lymph node. A dose-dependent facet existed for this response, as the increase in cell trafficking response persisted longer with 40 ìg or 400 ìg of plasmid-eGFP than with 4 ìg. This increased cell trafficking was independent of green fluorescent protein expression as both pCAN1-eGFP and pCAN1 induced similar responses.<p>Increased cell traffic induced by bacterial DNA was further characterized by determining whether bacterial DNA form was critical for this response. Treatment with intact plasmid (circular) DNA induced the greatest increase in cell traffic. In contrast, when plasmid DNA was digested with restriction enzymes into linear fragments of DNA, then cell trafficking was not significantly increased. <p>Numerous studies have shown that immunostimulatory guanosine-cytosine (CpG) motifs activate cells in vitro and can improve protective immune responses in vivo . Therefore we tested the hypothesis that the presence of immunostimulatory CpG motifs within plasmids was responsible for altering cell trafficking. It was shown that plasmids with added immunostimulatory CpG sequences affected cell trafficking in a dose-dependent manner. The injection of 40 ìg of pBISIA-88 caused an increase in cell trafficking while injecting 400 ìg pBISIA-88 failed to increase cell flow above control levels.<p>Analysis of cell populations collected in efferent lymph gives insight into treatment effects on immune surveillance. This is well-studied following treatment with antigen and in general, antigen induces a selective movement of cells into lymph that occurs 24 hours post-treatment. In contrast, plasmid DNA induced a rapid, non-selective movement of lymphocytes through the lymph node, suggesting that bacterial DNA and antigen may affect cell trafficking by different mechanisms and thereby have distinct effects on immune surveillance.<p>Short-term effects of bacterial DNA on lymph node architecture and cellular composition were also investigated to determine whether an increase in cell trafficking was associated with structural changes within the lymph node. While it was shown that bacterial DNA induced changes within a lymph node, including and increased number of germinal centers and an increased frequency of CD72*CD21* B cells, these changes were not correlated to increased cell trafficking.<p>In summary, bacterial DNA altered immune surveillance in sheep lymph nodes by changing cell traffic and lymph node architecture and composition. Many of these responses differed from responses observed by others following antigen treatment. The present observations suggest that bacterial DNA can function as an important signal to modulate immune surveillance and host responses to infections by pathogens.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Oncology. Title from pdf file main screen (viewed on October 24, 2009). Includes bibliographical references.

Η αντιμετώπιση του καρκίνου του προστάτη (αλλά και των υπόλοιπων νεοπλασματικών νοσημάτων) δεν περιλαμβάνει απλώς την ανεύρεση νέων χημειοθεραπευτικών φαρμάκων. Αφορά και την κατοχύρωση ορισμένων δεικτών που μπορούν να μας δώσουν περισσότερες πληροφορίες για την ύπαρξη της νόσου, αλλά και του σταδίου εξέλιξης που βρίσκεται έτσι ώστε να παίρνονται οι πλέον σωστές αποφάσεις για την διαχείριση του ασθενούς. Η βιβλιογραφική αναφορά που ακολουθεί θα ασχοληθεί εκτενώς με αυτά τα ζητήματα (δηλ. τους δείκτες νόσου/σταδίου νόσου του καρκίνου του προστάτη) και ιδιαίτερα με μια υποομάδα αυτών: των μοριακών δεικτών της σταδιοποίησης της λεμφαδενικής νόσου στον καρκίνο του προστάτη. Αφορά μια πολλά υποσχόμενη μερίδα μοριακών δεικτών στην αντιμετώπιση της συγκεκριμένης νόσου που ενδέχεται να επηρεάσει και την προσέγγιση άλλων συμπαγών όγκων. / Use of new molecular markers for staging of lymph node disease in the prostate cancer.

Orientadora: Rosemeri de Oliveira Vasconcelos / Banca: Valéria Marçal Felix de Lima / Banca: Antonio Carlos Alessi / Resumo: A Leishmaniose Visceral (LV) é uma zoonose de interesse em saúde pública e o cão é o principal reservatório doméstico de Leishmania chagasi. Este protozoário modula a resposta imune do hospedeiro. A citocina MIF favorece a permanência do macrófago no sítio da injúria e protege-o da apoptose. O objetivo deste estudo foi avaliar a presença de MIF nos linfonodos de cães com LV, comparando estes achados com a densidade de macrófagos parasitados nos linfonodos e com o estadiamento clínico dos cães. Neste estudo utilizou-se 33 cães oriundos de Araçatuba, município endêmico para a LV. Os cães foram distribuídos nos grupos assintomático (A), oligossintomático (O) e sintomático (S). As alterações morfológicas dos linfonodos poplíteo, subescapular, ilíaco e mesentérico foram avaliadas quanto ao perfil celular, determinado por escores. A carga parasitária e a imunodetecção de MIF foi feita pela técnica de imuno-histoquímica. Nos cães dos grupos com sinais clínicos (O e S), as reações inflamatória granulomatosa e plasmocitária foram predominantes. No grupo S, a atrofia linfóide predominou e associou-se ao maior número de granulomas e a maior carga parasitária. A densidade de parasitos nos linfonodos periféricos diferiu significativamente do grupo S para os demais grupos (P<0,05). A densidade de macrófagos imunomarcados com MIF foi maior no grupo S e apresentou uma correlação significativa com a carga parasitária no linfonodo poplíteo (P<0,05). Conclui-se que macrófagos são uma das células mais envolvidas na resposta ao parasito. O protozoário utiliza MIF para manter o macrófago no sítio de infecção, favorecendo a sua sobrevivência no hospedeiro / Abstract: Visceral leishmaniasis (VL) is a zoonotic disease critical for the public health and the dog is the main Leishmania chagasi„s domestic reservoir. This protozoan modulates the immune response of the host. The MIF cytokine facilitates the permanence of the macrophage in the site of injury and protects it from apoptosis. The aim of this study was to evaluate the MIF presence in the lymph nodes of dogs with VL, comparing these findings with the parasite density in the lymph nodes and with the clinical outcome of the dogs. Third three dogs from Araçatuba, an endemic city for VL, were used in this study. Dogs were distributed in the asymptomatic (A), oligosymptomatic (O) and symptomatic (S) groups. The cellular profile of the morphologic changes of the popliteal, subscapular, iliac and mesenteric lymph nodes were evaluated and scored. Immunohistochemistry did the parasite load and the MIF immunodetection technique. Granulomatous and plasmocitary inflammatory reaction predominated in the groups with clinical signs (O and S). In the S group, lymphoid atrophy predominated and was associated with high number of granulomas and high parasite load. Comparing the groups, the density of the parasites in the peripheral lymph nodes was significantly different for the S group (P<0.05). The density of the immunolabeling MIF macrophages was higher in the S group and had a significant positive correlation with the parasite load in the popliteal lymph node (P<0.05). In conclusion, the macrophages are the most involved cells, and the protozoan uses the MIF to keep the macrophage in the infection site, helping its survival in the host / Mestre

Made available in DSpace on 2015-06-17T19:33:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-28. Added 1 bitstream(s) on 2015-06-18T12:47:48Z : No. of bitstreams: 1 000823201.pdf: 2278739 bytes, checksum: eda9f8cb43d81ba5f142f83a5a6d1d39 (MD5)

Orientador: Andrigo Barboza De Nardi / Coorientador: Naida Cristina Borges / Banca: Manuela Cristina Vieira / Banca: Tiago Luís Eilers Treichel / Banca: Luís Gustavo Gosuen Gonçalves Dias / Banca: Annelise Carla Camplesi dos Santos / Resumo: O exame ultrassonográfico convencional tem baixa especificidade para detecção de alterações difusas, no entanto, o aumento da ecogenicidade renal é um dos achados mais comuns em cães e gatos com nefropatia. O objetivo deste estudo foi descrever a técnica de elastografia por irradiação da força de impulso acústico (ARFI) na avaliação dos rins de gatos adultos hígidos, e determinar os valores de referência. Foram incluídos neste trabalho dez gatos de pelo curto, adultos e saudáveis. Após sedação, foi realizada uma ampla tricotomia abdominal, para a realização do exame ultrassonográfico. O exame convencional, em Modo B foi realizado com um transdutor linear de 9,0 MHz usando equipamento de ultrassom ACUSON S2000 / SIEMENS (Siemens, Munique, Alemanha). A ecogenicidade, ecotextura, o tamanho e contornos renais foram avaliados. Com o mesmo transdutor, a elastografia foi realizada utilizando software para comparação das análises qualitativa e quantitativa do método. A elastografia qualitativa detectou a presença de deformidades e as velocidades de cisalhamento em diferentes locais (cranial, média e caudal) das regiões cortical e medular em ambos os rins. Os achados ultrassonográficos foram considerados normais em todos os gatos, sem alterações em arquitetura ou ecogenicidade renal. A elastografia qualitativa demonstrou que o córtex renal era deformável e apresentou áreas cinza escuro homogêneas; pelve renal tinha pouca rigidez (branca); e da medula mostrou um padrão de mosaico. Os r... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Conventional ultrasonography has low specificity to detect diffuse changes, however, increased renal echogenicity is one of the most common findings in dogs and cats with nephropathy. The objective of this study was to describe elastography by acoustic radiation force impulse (ARFI) in the assessment of adult cats' kidneys and determine reference values. Ten healthy adults short hair cats were included. After sedation, abdominal hair was clipped in order to perform ultrasound exams. The conventional examination (B-mode) was performed with a 9.0 MHz linear array transducer ultrasound equipment using ACUSON S2000/Siemens (Siemens, Munich, Germany). Echogenicity, echotexture, size and contours of each kidney were evaluated. With the same transducer, elastography was performed using a software that compares qualitative and quantitative analysis. Qualitative elastography detected deformities and shear wave velocity rates in different portions (cortical and medulla' cranial, media and caudal regions). The sonographic findings were normal in all cats, with no changes in renal architecture or echogenicity. Qualitative elastography showed that renal cortex was softer with dark gray homogeneous areas; renal pelvis presented lower stiffness (white); and medulla showed a mosaic pattern. Shear wave velocities were obtained in cranial, medial, and caudal regions of the cortex and medulla: 2.46 m/s ± 0.45m/s; 2,46m/s ± 0.48 m/ s 2,37 m/s ± 0, 42 m/s (P = 0.795) in the cortex and 1.61 m/s ± ... (Complete abstract click electronic access below) / Doutor