Philosophiae Doctor - PhD / Epidemiological studies have highlighted that leukaemia can be considered as the most prominent malignancy after radiation exposure during childhood. The lifetime risk on radiation-induced leukaemia for a given dose is 3 – 5 times higher for children compared to adults. The high risk at a young age is related to the elevated sensitivity of the red bone marrow where haematopoietic stem and progenitor cells (HSPCs) are located. HSPCs self-renewal capacity and long-life span increase their susceptibility to DNA damage accumulation, making them a major target of radiation-induced carcinogenesis. Proton beam therapy (PBT) is increasingly used to treat paediatric brain tumours due to its dose sparing properties compared to conventional X-ray based radiotherapy. However, concerns regarding the carcinogenic potential of secondary neutrons produced during PBT, especially in terms of their effect on HSPCs harboured in the cranial bone marrow of paediatric patients, remain. In this study, the radiobiological differences between 60Co γ-rays and p(66)/Be(40) neutron exposure was investigated to resolve the underlying mechanisms for the high radiosensitivity of HSPCs (CD34+ cells) isolated from umbilical cord blood (UCB). For both radiation qualities, an apparent dose-dependent increase in the frequency of radiation-induced MN was observed in CD34+ cells. Furthermore, increased cytogenetic damage was observed with the CBMN assay after neutron irradiation, which highlights its leukaemogenic potential. In addition, no difference was observed in the nuclear division index of the CD34+ cells post-irradiation between both radiation qualities. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX foci, 2 and 18 hours after radiation exposure. A significant higher number of DNA DSBs were observed 2 hours after neutron irradiation with 0.5 Gy, but decreased to similar levels for both radiation qualities after 18 hours. Different stages of apoptosis in CD34+ cells were studied at 18 and 42 hours numerous time points post-irradiation by flow cytometry using the Annexin/PI assay. In contrast to the γ-H2AX foci results, a significant difference in late apoptosis was observed at 18 hours and 42 hours between the two radiation qualities. The results point towards a fast error-prone DNA repair in HSPCs after neutron irradiation, which might contribute to genomic instability and leukemogenesis. In the second phase of the PhD project, the impact of age on radiosensitivity was investigated by comparing newborn T-lymphocytes with adult peripheral blood (APB) T-lymphocytes. The major difference between UCB and APB T-lymphocytes, is their immunophenotypic profile. Since it is known that different T-lymphocyte subsets have a difference in radiosensitivity, the fraction of CD4+, CD8+, naïve (CD45RA+) and memory (CD45RO+) T-lymphocytes was determined via flow cytometry in the two groups. The cytokinesis-block micronucleus (CBMN) assay was used to determine the extent to which age influences the frequency of cytogenic damage in response to 60Co γ-rays radiation. For both APB and UCB, an outspoken dose-dependent increase in the frequency of radiation-induced MN was observed at 0.5, 1, 3 and 4 Gy. However, no significant difference was observed at 4 Gy when comparing MN yields of APB and UCB. An increased radiosensitivity of newborn to adult donors of 34%, 42%, 29%, 26% and 16% was observed based on the MN scoring at doses of 0.5, 1, 2, 3 and 4 Gy, respectively. The lowest radiosensitivity was identified at the highest dose, which might explain the non-significant difference at 4 Gy. In addition, there was a clear trend that females were more sensitive to 60Co γ-rays radiation than males in both adults and newborns, even though the difference was not significant. The immunophenotypic study revealed that that both the CD4+ and CD8+ T-lymphocytes of newborns are mainly naïve. This is illustrated by the co-expression of CD45RA+ on 90.70% (range: 80.80% – 98.40%) and 95.90% (range: 89.60% – 98.80%) of CD4+ and CD8+ cells respectively. The composition of adult T-lymphocytes, in contrast, is clearly different with a more equal distribution between CD45RA+ and CD45RO+ subpopulations. This finding demonstrates that there are differences in the radiosensitivity between newborn and adult T-lymphocytes which might be linked to the immunophenotypic change of T-lymphocytes with age.

Para elucidar alguns aspectos da imunopatogenia da uveíte na erliquiose canina, avaliaram-se, por meio da análise imunistoquímica, bulbos oculares de cães experimentalmente infectados por Ehrlichia canis (grupo 1- G1), naturalmente infectados por Ehrlichia canis (grupo 2-G2) e na co-infecção natural de E. canis e Babesia sp. (grupo 3). Parâmetros clínicos e hematológicos foram avaliados. Empregaram-se o dot-blot-Elisa e a reação de imunofluorescência indireta para E. canis e Babesia sp. respectivamente. Para a confirmação diagnóstica, utilizou-se a reação de cadeia de polimerase (PCR) para E.canis. A contagem imunofenotípica para os anticorpos CD3, CD4, CD8, Tal1B5 e MAC 387 não demonstrou diferença significativa nas diferentes regiões analisadas do bulbo ocular. Observou-se no G1, G2 e G3, em todas as regiões analisadas diferença significativa da contagem imunofenotípica de células CD8+ em relação às células CD4+. Evidenciou-se diferença significativa entre a contagem percentual de células IgG2+ e CD79?+ na região de corpo ciliar do G3 em relação ao G1. A região da íris do G3, em relação ao G2, demonstrou diferenças significativas para o anticorpo IgG1. Evidenciou-se nos três grupos, a existência de correlação linear entre as células CD3+ e CD8+ e entre as células IgG2+ e CD79?+ em diversas regiões do bulbo ocular. O infiltrado inflamatório mostrou-se mais intenso nas regiões de corpo ciliar e ângulo iridocorneal, moderado em limbo e íris e mínimo em coróide. A avaliação semiquantitativa por score da intensidade do infiltrado inflamatório mostrou-se mais intensa nos animais que apresentavam co-infecção, sugerindo uma resposta imune mais intensa nesses cães. Demonstrou-se que o infiltrado inflamatório era composto, predominantemente, por linfócitos T CD3+ e B CD79+?. A maior porcentagem de células T CD3+ era CD8+, caracterizando, portanto, uma resposta imune do tipo citotóxica. A presença de células B CD79+? fala a favor de produção local de anticorpos. Observaram-se células imunomarcadas por IgG2 e poucas células marcadas por IgG1, sugerindo uma polarização da resposta imune para o padrão Th1, sendo um possível mecanismo imune de lesão na uveíte. Observou-se alta expressão de moléculas de MHC-classe II, sugerindo uma resposta imune intensa nos tecidos oculares, contudo ineficiente devido a deficiência de células CD4+. / The immunopathogenicity of uveitis in the canine ehrlichiosis was studied by conducting anatomy and immunohistochemical analyses in the ocular globes of dogs experimentally (Group 1) and naturally (Group 2) infected with Ehrlichia canis, and naturally coinfected with Ehrlichia canis and Babesia sp. (Group 3). Clinical and hematological parameters were evaluated. Dot-blot Elisa and indirect immunofluorescent test (IFA) were used to analyze E. canis and Babesia sp., respectively. PCR assay confirmed the diagnosis of the disease caused by E. canis. The immunophenotypic analysis with the antibodies CD3, CD4, CD8, Tal1B5 and MAC 387 revealed no significant differences between the various ocular regions analyzed. Significant differences were observed between the immunophenotypic analysis of CD8+ and CD4+cells from all regions analyzed from G1, G2 and G3; between the percentile counts of IgG2+ and CD79?+ in the ciliary body of G3 dogs when compared to G1, and for the IgG1 antibody counts of the iris region from G3 when compared to G2. A linear correlation between CD3+ and CD8+ cells and between IgG2+ and CD79?+ cells from several regions of the ocular globe was found for all three groups. The cellular inflammatory infiltrate observed in the ocular tissue was severe in the regions of the ciliary bodies and iridocorneal angle, moderate in the limbus and iris, and only slightly present in the choroids. A semiquantitative analysis of the intensity of the inflammatory infiltrate of the ocular globes was more intense in G3, which suggested a greater response of the immune system in these animals. The inflammatory infiltrate was composed of mainly B CD79+? and T CD3+ lymphocytes, which had CD8+ at a high percentage, thus characterizing this as a cytotoxic immune response. Results indicated that B CD79+? cells favored the production of local antibodies. IgG2 and very few IgG1 immunolabeled cells were found. This result indicated a polarization of the immune response to the Th1 pattern, which could be the mechanism of the lesions in the uveitis. A large number of cells expressing the MHC-class II molecules were observed, suggesting an intense immune response in the ocular tissues, however ineffective due to the CD4+ cell´s deficient.

Para elucidar alguns aspectos da imunopatogenia da uveíte na erliquiose canina, avaliaram-se, por meio da análise imunistoquímica, bulbos oculares de cães experimentalmente infectados por Ehrlichia canis (grupo 1- G1), naturalmente infectados por Ehrlichia canis (grupo 2-G2) e na co-infecção natural de E. canis e Babesia sp. (grupo 3). Parâmetros clínicos e hematológicos foram avaliados. Empregaram-se o dot-blot-Elisa e a reação de imunofluorescência indireta para E. canis e Babesia sp. respectivamente. Para a confirmação diagnóstica, utilizou-se a reação de cadeia de polimerase (PCR) para E.canis. A contagem imunofenotípica para os anticorpos CD3, CD4, CD8, Tal1B5 e MAC 387 não demonstrou diferença significativa nas diferentes regiões analisadas do bulbo ocular. Observou-se no G1, G2 e G3, em todas as regiões analisadas diferença significativa da contagem imunofenotípica de células CD8+ em relação às células CD4+. Evidenciou-se diferença significativa entre a contagem percentual de células IgG2+ e CD79?+ na região de corpo ciliar do G3 em relação ao G1. A região da íris do G3, em relação ao G2, demonstrou diferenças significativas para o anticorpo IgG1. Evidenciou-se nos três grupos, a existência de correlação linear entre as células CD3+ e CD8+ e entre as células IgG2+ e CD79?+ em diversas regiões do bulbo ocular. O infiltrado inflamatório mostrou-se mais intenso nas regiões de corpo ciliar e ângulo iridocorneal, moderado em limbo e íris e mínimo em coróide. A avaliação semiquantitativa por score da intensidade do infiltrado inflamatório mostrou-se mais intensa nos animais que apresentavam co-infecção, sugerindo uma resposta imune mais intensa nesses cães. Demonstrou-se que o infiltrado inflamatório era composto, predominantemente, por linfócitos T CD3+ e B CD79+?. A maior porcentagem de células T CD3+ era CD8+, caracterizando, portanto, uma resposta imune do tipo citotóxica. A presença de células B CD79+? fala a favor de produção local de anticorpos. Observaram-se células imunomarcadas por IgG2 e poucas células marcadas por IgG1, sugerindo uma polarização da resposta imune para o padrão Th1, sendo um possível mecanismo imune de lesão na uveíte. Observou-se alta expressão de moléculas de MHC-classe II, sugerindo uma resposta imune intensa nos tecidos oculares, contudo ineficiente devido a deficiência de células CD4+. / The immunopathogenicity of uveitis in the canine ehrlichiosis was studied by conducting anatomy and immunohistochemical analyses in the ocular globes of dogs experimentally (Group 1) and naturally (Group 2) infected with Ehrlichia canis, and naturally coinfected with Ehrlichia canis and Babesia sp. (Group 3). Clinical and hematological parameters were evaluated. Dot-blot Elisa and indirect immunofluorescent test (IFA) were used to analyze E. canis and Babesia sp., respectively. PCR assay confirmed the diagnosis of the disease caused by E. canis. The immunophenotypic analysis with the antibodies CD3, CD4, CD8, Tal1B5 and MAC 387 revealed no significant differences between the various ocular regions analyzed. Significant differences were observed between the immunophenotypic analysis of CD8+ and CD4+cells from all regions analyzed from G1, G2 and G3; between the percentile counts of IgG2+ and CD79?+ in the ciliary body of G3 dogs when compared to G1, and for the IgG1 antibody counts of the iris region from G3 when compared to G2. A linear correlation between CD3+ and CD8+ cells and between IgG2+ and CD79?+ cells from several regions of the ocular globe was found for all three groups. The cellular inflammatory infiltrate observed in the ocular tissue was severe in the regions of the ciliary bodies and iridocorneal angle, moderate in the limbus and iris, and only slightly present in the choroids. A semiquantitative analysis of the intensity of the inflammatory infiltrate of the ocular globes was more intense in G3, which suggested a greater response of the immune system in these animals. The inflammatory infiltrate was composed of mainly B CD79+? and T CD3+ lymphocytes, which had CD8+ at a high percentage, thus characterizing this as a cytotoxic immune response. Results indicated that B CD79+? cells favored the production of local antibodies. IgG2 and very few IgG1 immunolabeled cells were found. This result indicated a polarization of the immune response to the Th1 pattern, which could be the mechanism of the lesions in the uveitis. A large number of cells expressing the MHC-class II molecules were observed, suggesting an intense immune response in the ocular tissues, however ineffective due to the CD4+ cell´s deficient.

Philosophiae Doctor - PhD / The incidence of Diffuse large B cell Lymphoma has been increasing lately at an alarming rate especially, in developing countries like Sudan. The standard therapy in Sudan is based solely on the R-CHOP chemotherapy regimen, yet it has been noticed that Diffuse Large B cell Lymphoma prognosis remains unfavorable. The late diagnosis and the consequent side-effects of the therapy directly affected the disease’s poor outcome. There is a scarcity of scientific publications regarding DLBCL in Sudan, but the increased burden necessitates the need for further research.