Thesis (M.D. with Distinction in Research) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 52-62.

To supplement and modify the diagnosis and clinical research of B-cell Chronic Lymphocytic Leukemia (B-CLL), a new method based on cell imaging and image processing was developed and applied to the B-CLL patient samples. The fluorophore-labelled leukemia cells were clearly visualized, reflecting the positive/negative expression of the corresponding surface markers and their distribution. Computer algorithms were devised and used to analyze a large number of images. The fluorescence intensity of the labelled antibodies on a given cell directly reflects the expression of the corresponding surface markers. The morphology and size of leukemia cells were not identical even in the same patient’s sample and the size variation does not correlate with the number of surface markers. The amount of each surface marker was approximately fixed for each patient, but there were some relationships, for instance, the number of CD19 and CD38 markers were correlated to each other. The heterogeneous expression of surface markers confirmed an assumption that surface markers have their preferred membrane positions. One of the most important results is that the cell imaging and our image processing method has provided an alternative and reliable way to diagnose B-CLL and new insights in the prognosis of subtype of B-CLL.

A doença linfoproliferativa pós transplante (DLPT) é a proliferação tecidual secundária mais comum em crianças submetidas a transplante de órgãos sólidos, e representa um espectro de proliferação linfoide clínica e morfologicamente heterogêneo que vai desde uma hiperplasia policlonal indolente até linfomas agressivos. Aproximadamente 80% das DLPT estão associadas ao vírus Epstein Barr (EBV) e é originaria de células B, entre 10 a 15% tem origem em células T e aproximadamente 1% em células natural killer. O status sorológico negativo para EBV pré transplante e o grau de imunossupressão são os fatores de risco de maior relevância para o desenvolvimento desta enfermidade. A apresentação clínica é diversa e sintomas constitucionais podem estar presentes simulando infecção e ou rejeição ao órgão transplantado. A confirmação do diagnóstico por exame histopatológico é, habitualmente, necessária e a hibridização in situ geralmente detecta as partículas de EBV nos tecidos examinados. A melhor opção terapêutica ainda não está definida e atualmente o tratamento consiste na redução da imunossupressão associada ao uso do anticorpo Anti CD20 e ou quimioterapia citotóxica além da terapia celular disponível em alguns centros. Este estudo teve por objetivos avaliar a resposta tumoral ao uso do anticorpo Anti CD20 na DLPT de células B EBV positivas pós transplante de órgãos sólidos, além de associar a neoplasia à eventual inclusão genômica de DNA/EBV na célula neoplásica. Foram analisados retrospectivamente os prontuários de vinte e três pacientes com até 18 anos incompletos admitidos na Unidade de Internação do Serviço de Onco- Hematologia do Instituto da Criança (ICR) e Instituto do Tratamento do Câncer Infantil (ITACI) que desenvolveram DLPT CD20 positiva pós transplante de órgãos sólidos comprovada histologicamente entre 8 de março de 1995 e 13 de agosto de 2011. Todos foram submetidos à redução da imunossupressão, treze receberam Anti CD20 isolado, três Anti CD20 associado à quimioterapia citotóxica e sete pacientes não fizeram uso desta droga. A sobrevida global em dois anos dos pacientes que receberam Anti CD20 foi de 81,45% e quando comparada à sobrevida global de 37,5% dos que não receberam a droga revelou diferença estatística significativa (p=0,02). Todos os pacientes tiveram a detecção da proteína de latência viral de EBV Latent Membrane Protein1 (LMP1) na célula tumoral através da técnica de hibridização in situ realizada em blocos de parafina devidamente armazenados ao diagnóstico. A curta duração do tratamento com o Anti CD20, a toxicidade aceitável em relação às demais alternativas terapêuticas, a possibilidade de seu uso exclusivo, sua eficácia inclusive na doença de histologia agressiva e associação às demais alternativas de tratamento na doença refratária sugerem a inclusão desta droga no arsenal terapêutico atualmente disponível / Post-transplant lymphoproliferative disease (PTLD) is the most common secondary tissue proliferation that occurs in children after solid organ transplantation and represents a spectrum of clinical lymphoid proliferation and morphologic heterogeneity that goes from an indolent polyclonal hyperplasia to aggressive lymphomas. Approximately 80% of PTLD is associated with Epstein Barr virus (EBV) and is of B-cell origin, 10 to 15% of T-cells and approximately 1% of natural killer cells. EBV pretransplant seronegativity and the degree of immunosuppression are the most relevant risk factors for developing the disease. Clinical presentation is diverse and constitutional symptoms may simulate infection and/or organ transplanted rejection. Histopathologic examination is usually necessary to confirm diagnosis and, generally, in situ hybridization detects the EBV particles in examined tissues. The best treatment option is yet to be determined and the current treatment consists of immunosuppression reduction associated with the use of anti CD20 antibody and/or cytotoxic chemotherapy besides cell therapy only available in some centers. This study aimed to evaluate tumor response to the use of anti CD20 antibody in positive B-cell EBV PTLD after solid organ transplantation and the association of the neoplasia to the eventual inclusion of genomic EBV/DNA in the tumor cell. We retrospectively analyzed medical records of twenty-three patients under 18 years of age who were admitted to the inpatient unit of Serviço de Onco- Hematologia do Instituto da Criança (ICR) e Instituto do Tratamento do Câncer Infantil (ITACI) who developed histologically proven CD20 positive pediatric PTLD after solid organ transplantation between 8 March 1995 and 13 August 2011. All patients were submitted to immunosuppression reduction, thirteen received isolated Anti CD20, three Anti CD20 associated with cytotoxic chemotherapy and seven patients did not use this drug. The estimated 2-year overall survival rates of patients who received anti CD20 was 81.45% and when compared to the overall survival rates of those who did not receive the drug it was 37, 5%, showing a statistically significant difference (p = 0.02). All patients had the Epstein-Barr virus latency protein (latent membrane protein1 - LMP1) detected in tumor paraffin embedded stored at diagnosis by the in situ hybridization technic. The short duration of the Anti CD20 treatment, its acceptable toxicity compared to other therapeutic alternatives, the possibility of its exclusive use, its effectiveness in aggressive histology disease and the association with other treatment alternatives in refractory disease, suggest this drug inclusion to the currently available therapeutic arsenal

A doença linfoproliferativa pós transplante (DLPT) é a proliferação tecidual secundária mais comum em crianças submetidas a transplante de órgãos sólidos, e representa um espectro de proliferação linfoide clínica e morfologicamente heterogêneo que vai desde uma hiperplasia policlonal indolente até linfomas agressivos. Aproximadamente 80% das DLPT estão associadas ao vírus Epstein Barr (EBV) e é originaria de células B, entre 10 a 15% tem origem em células T e aproximadamente 1% em células natural killer. O status sorológico negativo para EBV pré transplante e o grau de imunossupressão são os fatores de risco de maior relevância para o desenvolvimento desta enfermidade. A apresentação clínica é diversa e sintomas constitucionais podem estar presentes simulando infecção e ou rejeição ao órgão transplantado. A confirmação do diagnóstico por exame histopatológico é, habitualmente, necessária e a hibridização in situ geralmente detecta as partículas de EBV nos tecidos examinados. A melhor opção terapêutica ainda não está definida e atualmente o tratamento consiste na redução da imunossupressão associada ao uso do anticorpo Anti CD20 e ou quimioterapia citotóxica além da terapia celular disponível em alguns centros. Este estudo teve por objetivos avaliar a resposta tumoral ao uso do anticorpo Anti CD20 na DLPT de células B EBV positivas pós transplante de órgãos sólidos, além de associar a neoplasia à eventual inclusão genômica de DNA/EBV na célula neoplásica. Foram analisados retrospectivamente os prontuários de vinte e três pacientes com até 18 anos incompletos admitidos na Unidade de Internação do Serviço de Onco- Hematologia do Instituto da Criança (ICR) e Instituto do Tratamento do Câncer Infantil (ITACI) que desenvolveram DLPT CD20 positiva pós transplante de órgãos sólidos comprovada histologicamente entre 8 de março de 1995 e 13 de agosto de 2011. Todos foram submetidos à redução da imunossupressão, treze receberam Anti CD20 isolado, três Anti CD20 associado à quimioterapia citotóxica e sete pacientes não fizeram uso desta droga. A sobrevida global em dois anos dos pacientes que receberam Anti CD20 foi de 81,45% e quando comparada à sobrevida global de 37,5% dos que não receberam a droga revelou diferença estatística significativa (p=0,02). Todos os pacientes tiveram a detecção da proteína de latência viral de EBV Latent Membrane Protein1 (LMP1) na célula tumoral através da técnica de hibridização in situ realizada em blocos de parafina devidamente armazenados ao diagnóstico. A curta duração do tratamento com o Anti CD20, a toxicidade aceitável em relação às demais alternativas terapêuticas, a possibilidade de seu uso exclusivo, sua eficácia inclusive na doença de histologia agressiva e associação às demais alternativas de tratamento na doença refratária sugerem a inclusão desta droga no arsenal terapêutico atualmente disponível / Post-transplant lymphoproliferative disease (PTLD) is the most common secondary tissue proliferation that occurs in children after solid organ transplantation and represents a spectrum of clinical lymphoid proliferation and morphologic heterogeneity that goes from an indolent polyclonal hyperplasia to aggressive lymphomas. Approximately 80% of PTLD is associated with Epstein Barr virus (EBV) and is of B-cell origin, 10 to 15% of T-cells and approximately 1% of natural killer cells. EBV pretransplant seronegativity and the degree of immunosuppression are the most relevant risk factors for developing the disease. Clinical presentation is diverse and constitutional symptoms may simulate infection and/or organ transplanted rejection. Histopathologic examination is usually necessary to confirm diagnosis and, generally, in situ hybridization detects the EBV particles in examined tissues. The best treatment option is yet to be determined and the current treatment consists of immunosuppression reduction associated with the use of anti CD20 antibody and/or cytotoxic chemotherapy besides cell therapy only available in some centers. This study aimed to evaluate tumor response to the use of anti CD20 antibody in positive B-cell EBV PTLD after solid organ transplantation and the association of the neoplasia to the eventual inclusion of genomic EBV/DNA in the tumor cell. We retrospectively analyzed medical records of twenty-three patients under 18 years of age who were admitted to the inpatient unit of Serviço de Onco- Hematologia do Instituto da Criança (ICR) e Instituto do Tratamento do Câncer Infantil (ITACI) who developed histologically proven CD20 positive pediatric PTLD after solid organ transplantation between 8 March 1995 and 13 August 2011. All patients were submitted to immunosuppression reduction, thirteen received isolated Anti CD20, three Anti CD20 associated with cytotoxic chemotherapy and seven patients did not use this drug. The estimated 2-year overall survival rates of patients who received anti CD20 was 81.45% and when compared to the overall survival rates of those who did not receive the drug it was 37, 5%, showing a statistically significant difference (p = 0.02). All patients had the Epstein-Barr virus latency protein (latent membrane protein1 - LMP1) detected in tumor paraffin embedded stored at diagnosis by the in situ hybridization technic. The short duration of the Anti CD20 treatment, its acceptable toxicity compared to other therapeutic alternatives, the possibility of its exclusive use, its effectiveness in aggressive histology disease and the association with other treatment alternatives in refractory disease, suggest this drug inclusion to the currently available therapeutic arsenal

No description available.

No description available.

Syfte: Att ta reda på hur relationen ser ut för B- respektive T-lymfocyter i biopsier av rotfyllda tänder med apikal parodontit med bedömd låggradig inflammation. Denna studie kommer även att analysera hur den inflammatoriska bilden ser ut i förhållande till beskrivna symtom i dessa biopsier. Material & metod: En pilotstudie utfördes på 10 biopsier från rotfyllda tänder med apikal parodontit med bedömd låggradig inflammation enligt Danesh et al 2019 klassificeringssystem. Biopsierna hämtades från Malmö universitets biobank. Immunhistokemisk infärgning av antigenerna CD20+ och CD3+ utfördes samt analyserades med hjälp av digitalmikroskop. Jämförelsen av symtomen från remisserna skedde efter att de histologiska resultaten sammanställts. Resultat: Det fanns fler T-lymfocyter än B-lymfocyter i 6 stycken av biopsierna. I de resterande 4 biopsierna var de lika många. I remisserna hade symtombilden inte angetts föralla biopsier. Slutsats: Enligt vår pilotstudie ser vi tendenser till att T-lymfocyter är fler än B-lymfocyter eller att de är lika många. Inga slutsatser kunde dras gällande symtombilden. Ett större material från olika remittenter krävs för definitiva slutsatser. En vidare infärgning av CD4+ samt CD8+ skulle vara intressant. / Aim: To investigate the relation between B- and T- lymphocytes in biopsies of root-filled teeth with apical periodontitis with assessed low-grade inflammation. This study will also analyse what the inflammation looks like in relation to the symptoms described in these biopsies.  Study design: A pilot study was performed on 10 biopsies of root-filled teeth with apical periodontitis with assessed low-grade inflammation according to the classification system of Danesh et al 2019. The biopsies were collected from Malmö University's biobank. An immunohistochemical staining of antigens CD20+ and CD3+ were performed and analysed by a digital microscope. A comparison of the symptoms from the referrals were performed once the histological results were compiled. Results: There were more T-lymphocytes than B-lymphocytes in 6 of the biopsies. In the remaining 4 biopsies there were an equal amount of B- and T-lymphocytes. The symptoms were not stated for all biopsies in the referrals.  Conclusion: According to our pilot study, we see tendencies that T lymphocytes are more than B lymphocytes or that they are equal. No conclusions could be drawn regarding the symptom picture. Larger material from different referrers is required for definitive conclusions. A further staining of CD4 + and CD8 + would be interesting.

Rituximab (IDEC C2B8) ist ein chimärer monoklonaler Antikörper, der gegen das CD20-Antigen auf B-Lymphozyten gerichtet ist. In klinischen Studien konnten Ansprechraten von 50 Prozent bei Patienten mit niedrigmalignen NHL erzielt werden [61, 62]. Bei den hochmalignen NHL waren die Ansprechraten geringer, doch auch hier konnte eine therapeutische Wirksamkeit von Rituximab nachgewiesen werden [108]. Der genaue Wirkungsmechanismus, durch welchen Rituximab seinen therapeutischen Effekt erzielt, ist weiterhin nicht vollständig geklärt. Hauptsächlich werden dabei Apoptose, Komplement-vermittelte zelluläre Zytotoxizität (CDC) und die Antikörper-vermittelte Zytotoxizität (ADCC) diskutiert. In der vorliegenden Arbeit wurde in vitro die Proliferationsinhibition und die Komplement-vermittelte zelluläre Zytotoxizität durch Rituximab an verschiedenen humanen B-Zellinien geprüft. Anschließend wurde die in vivo-Komplementaktivierung während einer Rituximab-Infusion untersucht. Es konnte gezeigt werden, dass Rituximab in vitro eine unterschiedliche Wirksamkeit bei verschiedenen Lymphomzellinien aufweist. Durch Zugabe von humanem Komplement konnte bei zwei Zellinien eine Rituximab-induzierte CDC beobachtet werden. Bei sechs von zehn Patienten mit unterschiedlichen NHL wurde in vivo ein Anstieg der C3a-desArg-Konzentration im Plasma beobachtet. Die Ergebnisse dieser Arbeit zeigen, dass das Komplementsystem ein wichtiger Mechanismus für die Wirkung von Rituximab zu sein scheint. Die klinischen Erfolge sind viel versprechend, zeigen jedoch auch, dass der therapeutische Nutzen von Rituximab als Monotherapie begrenzt ist. Derzeit laufende prospektive Studien untersuchen die Wirksamkeit von Rituximab in Kombination mit verschiedenen Chemotherapeutika [106, 107]. Erste Ergebnisse sind viel versprechend, doch es bleibt abzuwarten, ob sich diese Ergebnisse auch langfristig in einer Verbesserung der Überlebensrate widerspiegeln. / The chimeric anti-CD20 monoclonal antibody rituximab is a new therapeutic tool for treatment of relapsed B-cell lymphomas. Because rituximab mediates complement-dependent cellular cytotoxicity (CDCC) and antibody-dependent cellular cytotoxicity (ADCC) in the lymphoblastoid cell line SB, it is suggested, that these two mechanisms are responsible for the in vivo antilymphoma effect. We tested the antiproliferative effect of rituximab in 6 CD20 positive human B cell lymphoma cell lines. In the follicular lymphoma cell line, DOHH-2, 1 µg/ml rituximab induces an inhibition of proliferation of 75.5 % (n=5), and in the diffuse large cell lymphoma cell line, Daudi, an inhibition of proliferation of 27.3 % (n=5). No effect was seen in the prelymphocytic leukemia cell line, JVM-2, the hairy cell leukemia cell line, Bonna-12, or the two high-grade lymphoma cell lines, Granta-519 and Raji. To test, whether complement increases the effect of rituximab, we studied the combination of rituximab plus complement. Guinea pig complement (dilution 1:10) increases the inhibitory effect of rituximab on the proliferation of Daudi cells. The degradation product C3a(desArg) is produced during complement cascade activation and could be used as a sensitive and specific marker of complement activation in vivo. So, we measured plasma C3a(desArg) concentration in two CLL patients receiving rituximab monotherapy. In one patient, no increase of plasma C3a(desArg) concentration could be measured during infusion of rituximab. In the other patient, in two treatment cycles C3a(desArg) increases drastically after 2 h of rituximab infusion. We conclude differential in vitro and in vivo effects of rituximab on CD20 positive lymphoma cells. Rituximab inhibits in vitro the proliferation of follicular and diffuse large cell lymphoma cells, which could be amplified by complement in Daudi cells. Furthermore, rituximab mediates in vivo complement cascade activation, highly suggestive of in vivo CDCC.

OBJETIVOS: Correlacionar o nível de expressão das proteínas reguladoras do complemento (Cregs) CD55, CD59, CD35 e CD46 nos linfócitos B em uma coorte de pacientes com artrite reumatoide (AR) iniciando terapia com rituximabe (RTX) com a depleção e tempo de repopulação destas células no sangue periférico, associando, ainda, o nível de expressão destas proteínas à resposta clínica conforme os critérios do Colégio Americano de Reumatologia (ACR). MÉTODOS: Dez pacientes com AR receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. Análises imunofenotípicas para detecção de CD19, CD55, CD59, CD35 e CD46 foram realizadas pré-infusão e após 1, 2, 6, 12, 18 e 24 meses ou até recaída clínica. Depleção de linfócitos B no sangue periférico foi definida como valor de CD19 menor que 0,005x109/l no total de leucócitos. Resposta ACR20 em 6 meses foi considerada positiva e recaída clínica foi definida como perda dessa resposta. A não obtenção de ACR20 em 6 meses foi considerada falha de resposta ao tratamento. RESULTADOS: Dez mulheres com mediana de 49 anos e DAS28 basal de 5,6; nove delas soropositivas para fator reumatoide foram acompanhadas. Repopulação de linfócitos B ocorreu em 2 meses em cinco pacientes e em 6 meses nas demais. Houve correlação entre o nível de expressão basal de CD46 com o tempo de repopulação (coeficiente de correlação de -0,733, p=0,016). Tendência semelhante foi detectada com CD35, porém sem significância estatística (coeficiente de correlação de -0,522, p=0,12). Não houve associação entre resposta clínica e expressão das proteínas regulatórias do complemento. CONCLUSÕES: Expressão aumentada de CD46 foi preditora de repopulação mais precoce de linfócitos B em pacientes com AR tratados com RTX. Estudos com amostras maiores serão necessários para avaliar associação das demais Cregs. / OBJECTIVES: To correlate the level of expression of the complement regulatory proteins (Cregs) CD55, CD59, CD35, and CD46 on B cells from a cohort of 10 patients with rheumatoid arthritis (RA) initiating treatment with rituximab (RTX) with the depletion and time of repopulation of these cells in peripheral blood, additionally correlating the level of expression of these proteins to clinical response according to the criteria of the American College of Rheumatology (ACR). METHODS: Ten patients with RA received two 1g RTX infusions within 14 day intervals. Immunophenotype analyses for CD19, CD55, CD59, CD35 and CD46 were performed before the infusion and at 1, 2, 6, 12, 18 and 24 months or until recurrence. Depletion of B cells on peripheral blood was defined as the CD19 count < 0.005x109/l. ACR20 at 6 months was considered a good clinical response and recurrence was defined as loss of this response. RESULTS: Ten women with median age of 49 years and basal DAS28 of 5.6 were monitored; 9 were seropositive for rheumatoid factor. Repopulation of B cells occurred within 2 months in 5 patients and within 6 months in the remaining women. There was correlation between the basal level of CD46 expression and the time to achieve repopulation (correlation coefficient -0.733, p=0.016). A similar trend was observed with the CD35, but without statistical significance (correlation coefficient - 0.522, p=012). There was no association between clinical response and the complement regulatory proteins. CONCLUSIONS: Increased CD46 expression predicted earlier repopulation of B cells in RA patients treated with RTX. Studies with larger samples are necessary to assess the association with the other Cregs.

OBJETIVOS: Correlacionar o nível de expressão das proteínas reguladoras do complemento (Cregs) CD55, CD59, CD35 e CD46 nos linfócitos B em uma coorte de pacientes com artrite reumatoide (AR) iniciando terapia com rituximabe (RTX) com a depleção e tempo de repopulação destas células no sangue periférico, associando, ainda, o nível de expressão destas proteínas à resposta clínica conforme os critérios do Colégio Americano de Reumatologia (ACR). MÉTODOS: Dez pacientes com AR receberam duas infusões de RTX 1g separadas por intervalo de 14 dias. Análises imunofenotípicas para detecção de CD19, CD55, CD59, CD35 e CD46 foram realizadas pré-infusão e após 1, 2, 6, 12, 18 e 24 meses ou até recaída clínica. Depleção de linfócitos B no sangue periférico foi definida como valor de CD19 menor que 0,005x109/l no total de leucócitos. Resposta ACR20 em 6 meses foi considerada positiva e recaída clínica foi definida como perda dessa resposta. A não obtenção de ACR20 em 6 meses foi considerada falha de resposta ao tratamento. RESULTADOS: Dez mulheres com mediana de 49 anos e DAS28 basal de 5,6; nove delas soropositivas para fator reumatoide foram acompanhadas. Repopulação de linfócitos B ocorreu em 2 meses em cinco pacientes e em 6 meses nas demais. Houve correlação entre o nível de expressão basal de CD46 com o tempo de repopulação (coeficiente de correlação de -0,733, p=0,016). Tendência semelhante foi detectada com CD35, porém sem significância estatística (coeficiente de correlação de -0,522, p=0,12). Não houve associação entre resposta clínica e expressão das proteínas regulatórias do complemento. CONCLUSÕES: Expressão aumentada de CD46 foi preditora de repopulação mais precoce de linfócitos B em pacientes com AR tratados com RTX. Estudos com amostras maiores serão necessários para avaliar associação das demais Cregs. / OBJECTIVES: To correlate the level of expression of the complement regulatory proteins (Cregs) CD55, CD59, CD35, and CD46 on B cells from a cohort of 10 patients with rheumatoid arthritis (RA) initiating treatment with rituximab (RTX) with the depletion and time of repopulation of these cells in peripheral blood, additionally correlating the level of expression of these proteins to clinical response according to the criteria of the American College of Rheumatology (ACR). METHODS: Ten patients with RA received two 1g RTX infusions within 14 day intervals. Immunophenotype analyses for CD19, CD55, CD59, CD35 and CD46 were performed before the infusion and at 1, 2, 6, 12, 18 and 24 months or until recurrence. Depletion of B cells on peripheral blood was defined as the CD19 count < 0.005x109/l. ACR20 at 6 months was considered a good clinical response and recurrence was defined as loss of this response. RESULTS: Ten women with median age of 49 years and basal DAS28 of 5.6 were monitored; 9 were seropositive for rheumatoid factor. Repopulation of B cells occurred within 2 months in 5 patients and within 6 months in the remaining women. There was correlation between the basal level of CD46 expression and the time to achieve repopulation (correlation coefficient -0.733, p=0.016). A similar trend was observed with the CD35, but without statistical significance (correlation coefficient - 0.522, p=012). There was no association between clinical response and the complement regulatory proteins. CONCLUSIONS: Increased CD46 expression predicted earlier repopulation of B cells in RA patients treated with RTX. Studies with larger samples are necessary to assess the association with the other Cregs.