Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

Thesis (MMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Serum free light chains (FLC) are associated with imbalances in heavy and light chain production. Abnormal FLC ratios have been associated with risk of progression in certain diseases. Automated assays are available for their determination and they are used in the followup and management of patients with monoclonal gammopathies. Acceptable imprecision, specificity, accuracy and reproducibility between reagent batches is required to prevent under- or overestimation. Method validation is a standard process in every good laboratory to judge the acceptability of a new method. Reference intervals have been established in an older population, but it was considered important to verify these in our population. HIV is associated with B-cell dysfunction. As B-cell abnormalities are associated with disorders leading to monoclonal gammopathies, we postulated that the FLC levels and FLC ratio would be abnormal in HIV infected individuals. Methods and materials: Controls and pooled patient samples were used for the method validation study which included imprecision studies, linearity, recovery and interference studies, and method comparison studies, the latter compared our method to the same method used in another laboratory. For the reference interval study, blood was obtained from 120 healthy subjects. The following blood tests were performed: total protein, IgG, IgA, IgM, creatinine, protein electrophoresis, kappa FLC and lambda FLC. Using the kappa and lambda FLC results, a FLC ratio was determined. Three hundred and sixty-nine HIV positive subjects were then studied. The same tests were performed, as well as CD4+ counts and viral loads on the majority of them. Results: For the method validation study, precision, linearity and recovery was acceptable. Minimal interference was observed with haemolysis, lipaemia, bilirubin and rheumatoid factor. Our method showed comparable performance with the established method. For the reference interval study, all the creatinine values were normal, as were serum protein values. The serum protein electrophoreses were independently reviewed by 3 pathologists. Most were normal, with a few polyclonal increases seen, but no definite monoclonal bands. The 95% reference intervals for FLC’s as well as the FLC ratio were not statistically significantly different to the manufacturer’s recommendations. When examining the HIV positive study population, we found that FLC and FLC ratio were influenced by markers of HIV disease severity, such as CD4+ count, IgG, viral load, use of antiretroviral treatment and abnormal serum protein electrophoreses. Conclusion: The validation study of FLC showed excellent precision, acceptable bias, good linearity, good recovery and minimal interference, allowing routine introduction of the test. The 95% reference intervals obtained for our population were slightly higher than those recommended by the manufacturer. However, as most of the values fell within the manufacturer’s limits, we could accept the manufacturer’s recommended cut-offs. We found that FLC levels were definitely influenced by markers of HIV disease severity in our population and we postulate that they may be of use for follow-up of patients with HIV. / AFRIKAANSE OPSOMMING: Agtergrond: Serum vry ligte kettings (VLK) word geassosieer met ‘n wanbalans van ligte en swaar ketting produksie. Abnormale VLK ratios is geassosieer met ‘n risiko van verloop in sekere siektes. Geoutomatiseerde laboratorium toetse vir VLK is beskikbaar vir hul bepaling en word gebruik om pasiënte met monoklonale gammopatieë op te volg en te behandel. Aanvaarbare impresisie, spesifisiteit, akkuraatheid en herhaalbaarheid tussen reagens besendings is belangrik om onder- of oorbepaling te verhoed. Metode validasie is ’n standaard proses in elke goeie laboratorium om die aanvaarbaarheid van ’n nuwe metode te bepaal. Verwysingswaardes is al bepaal in ’n ouer populasie. Ons het besluit om die verwysingswaardes in ons populasie te bepaal. Mens-immuungebrekvirus (MIV) word geassosieer met B-sel disfunksie. Omdat B-sel abnormaliteite geassosieer word met afwykings wat tot monoklonale gammopatieë lei, het ons gepostuleer dat die VLK vlakke en VLK ratio abnormaal sal wees in MIV geïnfekteerde persone. Metodes en Materiale: Kontroles en pasiënt monsters is gebruik vir die metode validasie studie wat impresisie studies, lineariteit, herwinning, inmenging en metode korrelasie studies ingesluit het. In laasgenoemde geval is ons metode met dieselfde metode van ’n ander laboratorium vergelyk. Vir die verwysingswaardes studie is 120 gesonde persone se bloed gebruik. Die volgende toetse is bepaal: totale proteïen, IgG, IgA, IgM, kreatinien, proteïen elektroferese, kappa en lambda VLK. Die VLK ratio is bepaal deur die kappa en lambda resultate te gebruik. Driehonderd nege en sestig MIV-positiewe pasiente is gebruik vir die studie. Dieselfde toetse was gedoen, asook CD4+ tellings en virale ladings op die meerderheid van pasiente. Resultate: Vir die metode validasie studie, was presisie, lineariteit en herwinning aanvaarbaar. Minimale inmenging van hemolise, lipemie, bilirubien en rumatoïede factor is waargeneem. Ons metode het goed gekorreleer met die bepaalde metode. Die serum kreatinien en serum totale proteïen waardes was normaal tydens die verwysingswaardes studie. Die serum proteïen elektroferese was onafhanklik beoordeel deur 3 patoloë. Die meeste was normaal met enkele poliklonale verhogings, maar geen definitiewe monoklonale bande nie. Die 95% verwysings intervalle vir VLK en VLK ratio het nie statisties betekenisvol verskil van die vervaardiger se aanbevelings nie. In die studie van die MIV-positiewe studie populasie, het ons gevind dat VLK en VLK ratio beïnvloed word deur merkers van ernstige MIV siekte, soos CD4+ telling, IgG, virale lading, die gebruik van antiretrovale medikasie en abnormale serum proteïen elektroferese. Gevolgtrekking: Die validasie studie van VLK het uitstekende presisie, aanvaarbare partydigheid, goeie lineariteit, goeie herwinning en minimale inmenging gewys, wat die roetine instelling van die toets toegelaat het. Die 95% verwysingsintervalle wat vir ons populasie bepaal is, was effens hoër as die vervaardiger se aanbeveling. Die meeste van die waardes het egter binne die vervaardiger se limiete geval, dus kon ons die vervaardiger se afsnypunte aanvaar. Ons het gevind dat VLK vlakke definitief beïnvloed word deur merkers van die ernstigheidsgraad van MIV siekte in ons populasie en ons postuleer dat VLK van waarde kan wees met die opvolg van MIV pasiente. / NHLS / Harry Crossley for funding obtained

Die meisten Patienten mit hypertropher Kardiomyopathie und kongenitalen Herzerkrankungen exprimieren die atriale essentielle leichte Myosinkette (ALC-1) im Ventrikel, wo sie teilweise die ventrikuläre essentielle leichte Myosinkette (VLC-1) ersetzt. Diese VLC-1/ALC-1 Isoformveränderung korrelierte mit einem Anstieg der Zykluskinetik der Myosin-Querbrücken in chemisch gehäuteten Herzfasern aus hypertrophierten Humanventrikeln. Um die funktionelle Bedeutung der ALC-1 im gesamten intakten Herzen zu untersuchen, habe ich in der vorliegenden Arbeit ein transgenes Rattenmodell charakterisiert, das die humane ALC-1 (hALC-1) im Herzen exprimiert (TGR/hALC-1). WKY-Ratten dienten als genetisch korrekter Kontrollstamm. Mittels rekombinanter hALC-1 als Standard wurde die exprimierte hALC-1-Menge in SDS-Extrakten linker Ventrikel der TGR/hALC-1 im Western-Blot untersucht. 12 Wochen alte TGR/hALC-1 exprimierten etwa 17 mug hALC-1/mg SDS-Extrakt. Das exprimierte Transgen konnte in der Immunfluoreszenz zwischen den Z-Linien der Sarkomere lokalisiert werden. Die gerichtete Integrations des Transgens in das kardiale Myosinmolekül wurde zusätzlich noch in hochgereinigten Myosinpräparationen nachgewiesen. Analyse des linksventrikulären Proteoms durch 2D-PAGE, das zur Identifikation von etwa 3000 Proteinen führte, zeigte vergleichbare Proteinmuster in WKY und TGR/hALC-1. Die Untersuchungen der Kontraktilität des intakten isoliert perfundierten Herzen wurden mit Langendorff-Präparationen durchgeführt. Die Expression des hALC-1-Transgen führte zu statistisch signifikanten (p / Most patients with hypertrophic cardiomyopathy and congenital heart diseases express the atrial essential myosin light chains (ALC-1) in their ventricles, partially replacing the ventricular essential light chains (VLC-1). This VLC-1/ALC-1 isoform shift is correlated with an increase in cross-bridge cycling kinetics as measured using chemically skinned fibers from the hypertrophied ventricles of human hearts. To study the functional importance of hALC-1 in the whole intact perfused-heart, a transgenic rat model overexpressing hALC-1 (TGR/hALC-1) in the heart was generated. WKY rats were used as the respective genetically correct control strain. Using hALC-1HIST protein as a standard, the amount of transgenic protein expression was quantified by Immunoblot analysis of the left ventricular tissue extracts of the transgenic rats. Twelve-week-old TGR/hALC-1 expressed around 17mug hALC-1 per mg of whole SDS-soluble protein. The transgene was localized in-between the Z-lines of the sarcomere by immunofluoresecnce microscopy. Furthermore, the proper integration of the transgene into the rat ventricular myosin was confirmed by purifying myosin from rat ventricular tissues. Whole ventricular proteome analysis by 2D-PAGE, resolved approximately 3000 proteins spots in each TGR/hALC-1 and WKY animal. The whole protein expression patterns in both animal groups showed no differences with the exception of the transgenic hALC-1 protein spot. The perfused heart contractility parameters were evaluated using the Langendorff preparation. Expression of hALC-1 was accompanied by statistically significant improvements (p

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

No description available.

INTRODUÇÃO: O diagnóstico diferencial dos processos linfoproliferativos cutâneos de célula B permanece um desafio para patologistas, dermatologistas, hematologistas e oncologistas, apesar dos recentes avanços imunoistoquímicos e moleculares. OBJETIVO: Este trabalho avaliou o auxílio diagnóstico e as limitações da pesquisa da clonalidade utilizando a biologia molecular nos linfomas primários cutâneos de célula B e pseudolinfomas de células B, assim como a relevância da análise dos dados em conjunto com informações clínicas, histológicas e imunoistoquímicas. MÉTODOS: O estudo incluiu 31 casos de processos linfoproliferativos cutâneos de célula B classificados à histologia e imunoistoquímica como 14 linfomas, 6 pseudolinfomas e 11 casos inconclusivos. A pesquisa da clonalidade foi realizada em todos os casos por meio da pesquisa do rearranjo dos genes da cadeia leve kappa e pesada utilizando o método de PCR. RESULTADOS: Os resultados confirmaram monoclonalidade em 61,5% dos linfomas. Em adição, o método evidenciou monoclonalidade em 20% dos casos inconclusivos à avaliação histológica e imunoistoquímica. A pesquisa do rearranjo dos genes de cadeia leve kappa resultou mais contributiva em relação à pesquisa do rearranjo dos genes da cadeia pesada. CONCLUSÕES: Estes resultados demonstraram a utilidade do método no auxilio diagnóstico dos linfomas cutâneos. A maior contribuição no estudo da clonalidade dos processos linfoproliferativos cutâneos de células B, através da pesquisa do rearranjo dos genes de cadeia leve kappa em associação com a pesquisa do rearranjo dos genes de cadeia pesada, sugeriu a necessidade da utilização conjunta das duas técnicas para maior acurácia diagnóstica nestes casos. / INTRODUCTION: The differential diagnosis of the lymphoproliferative B-cell infiltrates remains an important challenge for pathologists, dermatologists, hematologists and oncologists, despite the recent advances in immunohistochemical and molecular techniques. OBJECTIVES: This study has evaluated the diagnostic aid and the limitations of the clonality analysis using molecular biology in cutaneous B-cell lymphomas and pseudolymphomas, as well as the relevance of this analysis when combined with clinical, histological and immunohistochemical data. METHODS: The study covered 31 cases of cutaneous lymphoproliferative B-cell infiltrates classified by histological and immunohistochemical characteristics as 14 lymphomas, 6 pseudolymphomas and 11 non-conclusive cases. The clonality analysis was performed in all cases using PCR to detect the pattern of immunoglobulin light kappa and heavy chains gene rearrangements. RESULTS: The results have confirmed monoclonality in 61,5% of lymphomas. In addition, the method showed monoclonality in 20% of the cases previously classified as a non-conclusive through histological and immunohistochemical evaluation. CONCLUSION: These results highlight the importance of the PCR clonality analysis as an ancillary diagnostic tool in cutaneous lymphoma. The research of the immunoglobulin light kappa gene rearrangement was more efficient resulting in a higher rate of monoclonality detection when compared to the heavy chain analysis. Nevertheless, the use of both protocols improves the sensitivity of the method.

INTRODUÇÃO: O diagnóstico diferencial dos processos linfoproliferativos cutâneos de célula B permanece um desafio para patologistas, dermatologistas, hematologistas e oncologistas, apesar dos recentes avanços imunoistoquímicos e moleculares. OBJETIVO: Este trabalho avaliou o auxílio diagnóstico e as limitações da pesquisa da clonalidade utilizando a biologia molecular nos linfomas primários cutâneos de célula B e pseudolinfomas de células B, assim como a relevância da análise dos dados em conjunto com informações clínicas, histológicas e imunoistoquímicas. MÉTODOS: O estudo incluiu 31 casos de processos linfoproliferativos cutâneos de célula B classificados à histologia e imunoistoquímica como 14 linfomas, 6 pseudolinfomas e 11 casos inconclusivos. A pesquisa da clonalidade foi realizada em todos os casos por meio da pesquisa do rearranjo dos genes da cadeia leve kappa e pesada utilizando o método de PCR. RESULTADOS: Os resultados confirmaram monoclonalidade em 61,5% dos linfomas. Em adição, o método evidenciou monoclonalidade em 20% dos casos inconclusivos à avaliação histológica e imunoistoquímica. A pesquisa do rearranjo dos genes de cadeia leve kappa resultou mais contributiva em relação à pesquisa do rearranjo dos genes da cadeia pesada. CONCLUSÕES: Estes resultados demonstraram a utilidade do método no auxilio diagnóstico dos linfomas cutâneos. A maior contribuição no estudo da clonalidade dos processos linfoproliferativos cutâneos de células B, através da pesquisa do rearranjo dos genes de cadeia leve kappa em associação com a pesquisa do rearranjo dos genes de cadeia pesada, sugeriu a necessidade da utilização conjunta das duas técnicas para maior acurácia diagnóstica nestes casos. / INTRODUCTION: The differential diagnosis of the lymphoproliferative B-cell infiltrates remains an important challenge for pathologists, dermatologists, hematologists and oncologists, despite the recent advances in immunohistochemical and molecular techniques. OBJECTIVES: This study has evaluated the diagnostic aid and the limitations of the clonality analysis using molecular biology in cutaneous B-cell lymphomas and pseudolymphomas, as well as the relevance of this analysis when combined with clinical, histological and immunohistochemical data. METHODS: The study covered 31 cases of cutaneous lymphoproliferative B-cell infiltrates classified by histological and immunohistochemical characteristics as 14 lymphomas, 6 pseudolymphomas and 11 non-conclusive cases. The clonality analysis was performed in all cases using PCR to detect the pattern of immunoglobulin light kappa and heavy chains gene rearrangements. RESULTS: The results have confirmed monoclonality in 61,5% of lymphomas. In addition, the method showed monoclonality in 20% of the cases previously classified as a non-conclusive through histological and immunohistochemical evaluation. CONCLUSION: These results highlight the importance of the PCR clonality analysis as an ancillary diagnostic tool in cutaneous lymphoma. The research of the immunoglobulin light kappa gene rearrangement was more efficient resulting in a higher rate of monoclonality detection when compared to the heavy chain analysis. Nevertheless, the use of both protocols improves the sensitivity of the method.