Despite improvements in therapy, the prognosis for non-small cell lung cancer (NSCLC) patients remains poor, and cure is only possible in localized tumors after surgical resection. A new generation of targeted cancer drugs has led to the expectation that lung cancer therapy can be significantly improved, but these drugs are today only an option in a small subset of NSCLC patients, and their effect is temporary. Therefore, the aim of this thesis was to characterize NSCLC in order to find new treatment targets and to evaluate biomarkers that further optimize therapy selection. In Paper I, the expression of the potential treatment targets claudin 6 and claudin 18.2 were evaluated based on immunohistochemical- and gene expression analysis. High ectopic protein and gene expression were demonstrated for both claudins in small subgroups of NSCLC. Clinical trials using humanized monoclonal antibodies against both proteins are ongoing in other cancer forms and may be extended to NSCLC. In Paper II, the prognostic impact of the inflammatory mediator cyclooxygenase 2 (COX-2) was evaluated. No prognostic significance was found in a meta-analysis incorporating gene expression data of 1337 NSCLC patients. Likewise, COX-2 protein expression in tumor cells was not associated with survival in two independent NSCLC cohorts. However, in one of the analyzed cohorts, higher COX-2 expression in the tumor stroma was associated with longer survival and may therefore be a subject for further investigation. In Paper III, tumor and stromal COX-2 protein expression was examined in patients treated with the COX-2 inhibitor celecoxib in order to evaluate if COX-2 expression is a predictive biomarker for benefit of celecoxib therapy. Celecoxib did not prolong overall survival neither in the whole cohort nor in patients stratified according to COX-2 expression in tumor or stromal cells. Noteworthy, a tendency towards longer survival was again demonstrated in patients with high COX-2 stromal expression. In Paper IV, the diagnostic methods for identification of ALK rearrangements were assessed in a large representative Swedish NSCLC population. Fluorescence in situ hybridization (FISH), as the diagnostic standard, was compared to two immunohistochemical assays. ALK gene expression levels were incorporated to supplement the molecular data. The frequency of ALK rearrangements was lower than previously reported. The different methods to detect the ALK fusion demonstrated overlapping results. However, the overlap was poor, so the methods cannot be regarded as interchangeable and should thereby be interpreted with caution when used in clinical diagnostics. In summary, this thesis applied an integrative translational approach to characterize potential new treatment targets and to evaluate the detection of existing predictive biomarkers in NSCLC.

Intrauterine Bälle („small uterine device“; IUDs) sind aus Glas, Plastik oder Metall und wer-den vaginal in den Uterus eingeführt. Bei Stuten werden IUDs im Turniersport zur Unterdrü-ckung der unerwünschten Verhaltensänderungen während der Rosse eingesetzt. Der Einsatz ist einfach, günstig und minimal invasiv. Der Effekt wird durch eine Verlängerung der primären lutealen Phase erzielt, wobei jedoch der exakte Ablauf des Wirkmechanismus bisher ungeklärt ist. Hypothesen wie eine Scheinträchtigkeit, ein Placeboeffekt bei den Besitzern und eine chronische Endometritis werden in der Literatur diskutiert. Für die Untersuchung wurden 30 Stuten in vier Gruppen (G) eingeteilt: G1: KB (künstlich besamt) und tragend (n=8); G2: KB, nicht-tragend (zyklisch; n=7); G3: IUD, verlängerte luteale Phase (n=7) IUD-P (IUD-Positiv); G4: IUD, reguläre luteale Phase (n=8) IUD-N (IUD-Negativ). Die Uterusbiospien wurden am Tag 15 post ovulationem entnommen. Das Ziel der Studie war, mittels immunhistologischer Untersuchungen die Expression des Enzyms Cyclooxygenase 2 (COX2), verschiedener uteriner Proteine (Uteroglobin, Uterofer-rin, Uterokalin), Hormonrezeptoren (Östrogen-, Progesteronrezeptor) und des Proliferations-markers Ki-67 Antigen in endometrialen Biopsien bei IUD-Stuten darzustellen sowie die erhobenen Befunde mit den Ergebnissen am equinen Endometrium künstlich besamter Stuten zu vergleichen. Weiter wurde in den Endometriumbioptaten das Auftreten von Entzündungszellen analysiert (neutrophile und eosinophile Granulozyten, Mastzellen, Lymphozyten, Plasmazellen und Makrophagen). Die statistische Auswertung erfolgte mittels SPSS (SPSS Software-GmbH München). Hinsichtlich der Altersverteilung sind die Stuten der Gruppe 1 (KB/tragend) jünger als Tiere der Gruppe 2 (KB/nicht-tragend). Ein entsprechendes Ergebnis kann für die Stuten der Grup-pe 3 (IUD-P) im Vergleich zu den Tieren der Gruppe 4 (IUD-N) erhoben werden. Darüber hinaus sind Angiopathien bei den Pferden der Gruppe 1 bzw. 3 geringer ausgeprägt als bei den Stuten der Gruppe 2 bzw. 4. Immunhistologisch sind die endometrialen Drüsenzellen der Tiere aus Gruppe 1 durch eine maximale Uterokalin-(UK)-Expression gekennzeichnet, wohingegen Uteroferrin (UF) ledig-lich schwach exprimiert wird. Eine COX2-Expression kann bei diesen Stuten nicht beobachtet werden. Im Vergleich zu den graviden Pferden (Gruppe 1) zeigen künstlich besamte, nicht-tragende Stuten (Gruppe 2) zwar eine ausgeprägte UF- und COX2-Expression, UK wird dagegen lediglich gering exprimiert. Die KB-tragenden (Gruppe 1) und nicht-tragenden (Gruppe 2) Tiere sind somit durch eine ihrem Reproduktionszyklus entsprechende Expression der genannten Marker gekennzeichnet. Die IUD-Stuten (Gruppe 3 und 4) dagegen zeigen eine variable COX2, UF- und UK-Expression. Keine statistisch signifikanten Unterschiede sind zwischen allen Gruppen in der UG- und der Ki-67 Antigen-Expression nachweisbar. Stuten mit einer verlängerten lutealen Phase (Gruppe 1 und 3) und hohen Progesteronwerten im Serum besitzen eine geringere ER- und PR-Expression als die Versuchstiere mit einer regulären lutealen Phase (Gruppe 2 und 4) und einer Östrogendominanz. Die Auszählung der Entzündungszellen zeigt keine statistisch signifikanten Unterschiede hinsichtlich der Anzahl von neutrophilen und eosinophilen Granulozyten, Mastzellen, Plasmazellen sowie Lymphozyten zwischen den Gruppen. Im Stratum compactum ist die Zahl der Makrophagen in Gruppe 1 (tragend) signifikant höher als in Gruppe 2 (zyklisch), ebenfalls zeigt sich ein Anstieg dieser Zellen von der Gruppe 4 zu 3, allerdings ist diese Erhöhung nicht statistisch signifikant. Ein Placeboeffekt bei den Besitzern kann nahezu ausgeschlossen werden, da die Expression der Proteine (UF, UK) und COX2 in den IUD-Stuten, im Vergleich zu den KB-Stuten, signi-fikante Unterschiede aufweist. Da im eigenen Untersuchungsgut zwischen den tragenden Stuten (Gruppe 1) und den IUD-Stuten der Gruppe 3 (vlP)/“Scheinträchtigkeit“ ein signifikant unterschiedliches Expressionsverhalten hinsichtlich des Enzyms COX2 und des Proteins UF besteht, kann die Hypothese einer Scheinträchtigkeit ebenso als unwahrscheinlich eingestuft werden. Bei keiner der IUD-Stuten konnte eine chronische Endometritis nachgewiesen werden, somit ist auch diese Hypothese als Ursache des IUD-Effektes eher unwahrscheinlich. Die erhöhte Anzahl an Makrophagen in der Gruppe 3 könnte jedoch hinweisend auf einen lokalen Effekt der IUDs im Sinne einer Fremdkörperreaktion sein. Da hinsichtlich des Auftretens einer Endometritis zwischen resistenten und empfänglichen Tieren unterschieden wird, könnte somit die erhöhte Anzahl an Makrophagen in der Gruppe 3 möglicherweise als Hinweis auf „Endometritis empfängliche Stuten“ gewertet werden und dies eine Ursache für die variierende Wirksamkeit der Rosseunterdrückung mittels IUDs darstellen. Die eigenen Untersuchungen zeigen ferner, dass die IUD-Stuten mit verlängerter lutealer Phase (IUD-positive Wirkung, Gruppe 3) jünger sind im Vergleich zu den Tieren, die trotz IUD eine Luteolyse (IUD-negative Wirkung, Gruppe 4) aufweisen. Zudem weisen die IUD-P Tiere geringere An-giopathien auf. Möglicherweise sind zudem das Alter und die Perfusion des Uterus bedeutend für die Wirksamkeit der IUDs in Equiden. Abschließend kann somit der Wirkmechanismus der IUDs auch mittels der durchgeführten Untersuchungen nicht endgültig geklärt werden. Unter Berücksichtigung der erhobenen Be-funde sollten zukünftige Studien insbesondere Untersuchungen zu Entzündungsmediatoren, wie z.B. Zytokine, beinhalten.

<p>In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function.</p><p>To analyze protein expression in <i>in vitro</i> cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells <i>in vivo</i>. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types. </p><p>Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas.</p><p>In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.</p>

Breast cancer (BC) is one of the leading causes of cancer-related deaths in Canadian women. Aggressive BCs (e.g. triple-negative subtype; TN) present a clinical challenge as defined biomarkers, particularly those indicative of unique cancer-associated signaling pathways, are needed to improve prognostication and prediction of therapeutic response. Overexpression of Src and its substrates, Ezrin and Tks5, have been associated with poor prognosis in many cancers. We have previously shown that Ezrin regulates proteolytic-independent invasion, while others have shown that Tks5 is associated with proteolytic-dependent invasion. Thus, expression of Ezrin versus Tks5 in BC cases may represent different invasion modalities. Additionally, immunofluorescence (IF)-based technologies may provide a more quantitative and objective approach for analysis of biomarker expression and subcellular compartmentalization compared to immunohistochemistry (IHC). In this study, I hypothesize that expression and subcellular localization of Src, Ezrin and Tks5, have improved prognostic significance in BCs, compared to current clinico-pathological parameters. To assess this, I optimized an IF-based automated quantification analysis (AQUA) system to measure subcellular expression in a 63-patient BC cohort and tested associations with clinico-pathological data. This thesis presents that: 1) Expression of Src and Ezrin increased, but that of Tks5 decreased in breast tumours compared to normal breast. 2) Src and Ezrin localized to the apical regions of normal breast epithelia but shifted to the cytoplasm in breast tumours. Tks5 exhibited a granular basal expression in normal breast epithelia, and is weakly expressed in tumour cellular compartments. 3) In our 63-patient cohort, Src and Ezrin had significant correlations with multiple clinico-pathological parameters, including TN status and lymphovascular invasion. 4) Clinico-pathological associations with IF-based AQUA scoring are directly comparable to conventional manual IHC scoring. Our study supports the role of Src and Ezrin as potential prognostic biomarkers for BC. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-07-09 12:51:09.527

Early pregnancy is characterized by complex interactions between blood vessels, leukocytes, and conceptus-derived trophoblasts within the gestational uterus. Uterine Natural Killer (uNK) cells become the most abundant leukocyte during decidualization and produce a wide array of angiogenic factors, yet little is known regarding their early pregnancy functions. To characterize the role(s) of uNK cells, whole mount in situ immunohistochemistry of live early implant sites was performed. A timecourse examination of murine early pregnancy (virgin, and gd4.5-9.5) implantation sites was performed. Comparison of Gd6.5, 8.5 and 9.5 implant sites from BALB/c+/+ controls (BALB/c) and BALB/c-Rag2-/-Il2rg-/- (alymphoid) identified anomalies that result from the absence of lymphocytes. In alymphoid decidua basalis, mesometrial angiogenesis was widespread but pruning of nascent vessels within alymphoid decidua basalis was deficient. As early gestation progressed, vessels of alymphoid decidua basalis showed no evidence for remodeling. Alymphoid implantation sites showed ~24h delay in uterine lumen closure and embryonic development. To determine if uNK cells would normalize the anomalies observed in alymphoid implantation sites, adoptive cell transfer of NK+ B- T- marrow to alymphoid mice was performed. All of the above anomalies were reversed by adoptive transfer of NK+B-T- marrow. My results suggest that uNK cells support vascular growth and development which ensures the decidua can support the growing conceptus early in pregnancy prior to formation and function of the placenta. Human decidual NK cells may fill similar roles and be important targets for strategies designed to correct intra-uterine growth restriction. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2013-08-02 08:42:06.487

Thesis (PhD)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: BACKGROUND Lymphomas arise in cells of the immune system. They vary widely in cytomorphology, immunophenotype and clinical course as well as response to treatment - all of which are factors that determine prognosis. The substantial geographical differences that exist for Hodgkin and other lymphoproliferative disorders have been previously reported from the Lymphoma Reclassification Project. OBJECTIVE This investigation was in two parts. In the first we tested the hypothesis that, using comparable treatment regimens, outcome from a private academic centre in the Western Cape region of South Africa would be similar to that from the first world. To this end a series of 512 individuals were analysed. In the second tissue samples from these patients where the most common subtype is diffuse large B-cell lymphoma (n=93) randomly receiving either standard combination chemotherapy in the form of the CHOP regimen or the identical program with rituximab which is an anti-CD20 monoclonal antibody were further investigated in cooperation with the University of Nebraska Medical Centre using an immunohistochemistry based method, also known as tissue microarray. PATIENTS AND METHODS In the first cohort consecutive and comprehensive records of patients diagnosed with lymphoma aged 14 years and older seen between October 1998 in July 2006, were scrutinized. After exclusion for a variety of reasons 398 cases suitable for further definitive study remained. In the second group tissue samples from a total of 149 biopsy proven de novo diffuse large Bcell variants could be further evaluated. After additional refinement a total of 93 remained that met all the entry criteria for the study in which 48 received chemotherapy alone and the remaining 45 chemo-immunotherapy. Demographic features were well matched. The initial stratification of these 93 cases, based on phenotyping with immunohistochemistry employing a tissue microarray method, yielded two populations depending on the criteria used. Each of these primary subdivisions was further evaluated for expression of BCL2 and LMO2 both of which are recognised to predict response. Finally, for each variable, clinical characteristics and survival outcome were compared between chemotherapy as a single modality or the same regimen combined with rituximab. STATISTICAL ANALYSIS Overall and event-free survival were calculated as the years from diagnosis to death, loss to follow-up, first progression or relapse respectively. The Kaplan Meier method was used to estimate distribution and the log rank test to compare survival between groups. Patient characteristics for each cohort specified were tested with Chi-squared or Fisher's exact test for small samples. RESULTS In the first part of this study all 398 cases were retrospectively analysed and showed a similar treatment outcome with regards to overall and event-free survival at 36 months when compared to first world reference centers. Adverse factors identified were similar to published experience comprising constitutional symptoms, prior treatment with chemotherapy, intermediate or high-risk scores as defined by the International Prognostic Index, histologic grading and certain anatomical sites of primary tumour. In contrast gender, staging by Rye or Rai classification, retroviral infection and prior treatment with radiotherapy were without effect. In the second part tissue samples from 93 de novo DLBCL, subtyped using an investigational immunohistochemical based Tissue Micro Array (TMA) were contrasted to the approximately corresponding categories as defined either by Hans and associates using a three marker panel into germinal or non-germinal centre subtypes or by Choi and colleagues with two additional antibodies into germinal centre or activated B-cells. Not surprisingly when compared to DNA-based gene expression profiling, as a reference point, concordance was different being 86 as opposed to 93% for the respective algorithms. The rationale for this exercise was to determine relative cost-effectiveness in an affordable but accurate technology that may be applicable to under resourced areas of the globe. Additionally the prognostic marker expression for BCL2 and LMO2 was investigated with regards to treatment outcome. Using the investigational tissue microarray approach the addition of rituximab to standard chemotherapy group did not show any significant improvement on 5 years overall (63% vs 59%, p 0.68) or event-free survival (42% vs 39%, p 0.94). Similarly no differences were evident in subtype analysis. Interestingly however, when segregated on the Choi criteria, cytotoxic drugs alone showed a non-significant trend in improved survival (74% vs 55%, p 0.32) as well as event-free survival (44% vs 40%, p 0.42) for the germinal centre as opposed to the activated B-cell subtype. Nevertheless not even a small difference could be demonstrated in the presence of rituximab. According to Choi, both regimens (chemotherapy with or without the addition of rituximab) revealed similar results to the Hans algorithm on 5 years OS as well as 3 year EFS when comparing GCB versus non-GCB subgroups. BCL2 and LMO2 marker expression of the respective immunohistochemical (IHC) subtype, despite small sample size, revealed the following. Analysis by Choi criteria on survival for BCL2, no matter for which subsets (GCB or ABC) or treatment modality (chemotherapy with or without the addition of rituximab) showed no difference in 5 years OS or EFS. However, in contrast, a significant difference for better EFS (p=0.0015) in the BCL2 positive group of the ABC subgroups subtypes treated with rituximab containing chemotherapy. These results must be interpreted with care due to the very low sample size and the follow up time of only 9 months. For LMO2 similar results on survival outcome were seen thus showing no difference in 5 years OS or EFS – regardless of subtype or treatment modality. Also here, this was contrasted by better EFS (p=0.039) in the LMO2 positive group of ABC subtypes when treated with the rituximab containing regimen. Again the reservation about small numbers and the 14 months observation period apply. DISCUSSION AND CONCLUSION Most of the studies examining the proportions of subtypes in large series of DLBCL patients have been predominantly carried out on Western populations. While 50% of patients in North America or Europe express GCB phenotypes, this is only 31% in their Asian counterparts. Thus far, no study has been published on lymphoma subtypes in South African populations using a Tissue Micro Array (TMA) method. Despite certain limitations of this study, due to a variety of reasons such as loss of analysable data, variability of representation of the South African population as a whole or low sample size leading to a potential source of error, these results confirm that lymphoproliferative disorders are heterogeneous. Neverless this group can be treated successfully if exact staging, classification and risk assessment defines the holistic management plan – no matter if in a developed or under resourced setting. With the introduction of sophisticated methods such as gene expression profiling (GEP), diffuse large B-cell lymphoma (DLBCL) can be classified into the prognostically favourable germinal center B-cell–like (GCB) and the more aggressive activated B-cell–like (ABC) subtypes. Against the background of resource restriction we therefore used an immunohistochemical (IHC) stain method to analyse formalin-fixed, paraffin-embedded tissue samples. Here the algorithm by Choi et al., originally described by Hans et al., showing a high concordance rate, was comparable to the GEP classification. The use of the IHC based TMA methodology was shown to be a simple, cost effective and a robust alternative to GEP which is currently regarded as the gold standard for the classification in lymphomas. It provides a useful prognostic tool in stratifying DLBCL or other entities in future, even when frozen tissue samples are not available for GEP analysis. With the current budgetary limitations in public hospitals chemotherapy protocols for lymphoproliferative disorders exclude agents such as rituximab. Local therapeutic drug committees consider the approximately 15% overall survival benefit seen at 5 years for DLBCL when rituximab is added to combination chemotherapy as too marginal for justifying the arising additional expenses. Accordingly, demonstration that a specific molecular subtype accounts for superior outcome when using these regimens is needed and would provide convincing evidence for the use of this monoclonal antibody in a resource constrained setting. / AFRIKAANSE OPSOMMING: AGTERGROND Limfome ontstaan vanuit selle van die immuunsisteem. Hulle toon ‘n groot verskil met betrekking tot sitomorfologie, immunofenotipe, kliniese verloop, asook respons op behandeling – almal faktore wat prognose bepaal. Die Limfoom Herklassifikasieprojek het getoon dat daar substansiële geografiese verskille bestaan vir Hodgkin se limfoom en ander limfoproliferatiewe toestande. Huidige navorsing oor hierdie maligniteite skep die indruk dat hulle skaars is in Afrika, met die inligting wat wel beskikbaar is hoofsaaklik gebaseer op gevallestudies deur klinici en patoloë. DOEL Hierdie studie bestaan uit twee dele. In die eerste toets ons die hipotese dat die uitkomste vir die behandeling van limfoom in ‘n privaat akademiese instansie geleë in die Wes-Kaap, Suid- Afrika, dieselfde is as wat in die res van die wêreld gesien word indien soortgelyke behandeling toedgedien word.`n Reeks van 512 pasiënte is in die analise gebruik. Vir die tweede hipotese het`n kohort van die mees algemene subtipe, naamlik diffuse groot B-sellimfoom (DLBCL) (n = 93), lukraak òf die standaardkombinasie-chemoterapie in die vorm van CHOP òf `n identiese program met rituximab, `n anti-CD20 monoklonale teenliggaam, gekry. Die teenliggaam is in samewerking met die Universiteit van Nebraska se Mediese Sentrum getoets met behulp van `n immunohistochemies-gebaseerde metode, ook bekend as weefsel mikro-skikking (tissue microarray or TMA). PASIËNTE EN METODE Vir die eerste kohort is die opeenvolgende en volledige rekords van pasiënte wat ouer as 14 was en tussen Oktober 1998 en Julie 2006 in `n privaat-gebaseerde akademiese instansie gediagnoseer is, noukeurig ondersoek. Na sekere uitsluitingskriteria toegepas is, was daar 398 gevalle wat geskik was vir verdere analise. In die tweede deel van die navorsing kon 149 pasiënte wat nuut met diffuse groot B-sel-limfoom (DLBCL) gediagnoseer is, verder geëvalueer word. Drie-en-neëntig van hierdie 149 pasiënte het aan die kriteria vir insluiting voldoen; 48 het slegs die standaard chemoterapie (CHOP) ontvang en die oorblywende 45 het chemo-immunoterapie (chemoterapie plus rituximab (R-CHOP) ontvang. Die demografiese eienskappe van beide groepe het goed vergelyk. Vanweë die kriteria wat gebruik is, het die aanvanklike stratifikasie van 93 gevalle, wat deur middel van die TMA-metode op fenotipering met immunohistochemie gebaseer was, twee populasies gelewer. Hierdie primêre subdivisies is verder geëvalueer vir uitdrukking van BCL2 en LMO2, beide erkende voorspellers van respons. Laastens is kliniese eienskappe en oorlewingsuitkoms ná behandeling met chemoterapie as `n enkel modaliteit of dieselfde chemoterapie met rituximab, vir elke veranderlike met mekaar vergelyk. STATISTIESE ANALISE Algehele en insident-vrye oorlewing is bereken as die jare vanaf diagnose tot sterfte, verdwyning van pasiënte tydens opvolg, eerste progressie of terugval van die toestand. Die Kaplan Meier-metode is gebruik om distribusie te bepaal en die log rank-toets om oorlewing tussen die groepe te vergelyk. Pasiëntkenmerke vir elke gespesifiseerde kohort is met die Chikwadraattoets of Fisher se eksakte toets in die geval van kleiner groepe getoets. RESULTATE In die eerste gedeelte van die studie is al 398 gevalle terugwerkend geanaliseer en daar is gevind dat die behandelingsuitkoms met betrekking tot totale oorlewing en insident-vrye oorlewing teen 36 maande vergelykbaar was met uitkomste in eerstewêreldsentrums. Nadelige faktore met betrekking tot oorlewing was soortgelyk aan reeds gepubliseerde data en het die volgende ingesluit: gestelsimptome, voorafgaande behandeling met chemoterapie, intermediêre of hoë-risiko tellings soos deur die Internasionale Prognostiese Indeks bepaal, histologiese gradering en sekere anatomiese setels van primêre tumor. In teenstelling met internasionale ondervinding het geslag, steiering volgens Rye- of Rai-klassifikasie, retrovirale status en vorige behandeling met radioterapie geen invloed gehad nie. In die tweede gedeelte is weefsel van 93 nuut-gediagnoseerde DLBCL-pasiënte wat deur middel van die TMA-metode subtipeer is, vergelyk met naastenby ooreenstemmende kategorieë soos deur Hans et al. met `n drie-merkerpaneel in kiem- en nie-kiemsentrumsubtipes, of deur Choi et al. met twee bykomende teenliggame in kiemsentrum of geaktiveerde B-selle gedefinieer. Met die DNA-gebaseerde geen-uitdrukkingsprofiel as `n verwysingspunt, was die ooreenstemming tussen die onderskeie algoritmes na verwagting verskillend, met 86% teenoor 93%. Die onderliggende rasionaal was om die relatiewe lonendheid te bepaal van `n bekostigbare maar akkurate tegnologie, wat moontlik in gebiede met onvoldoende hulpbronne toegepas kon word Verder is die prognostiese merkeruitdrukking vir BCL2 en LMO2 ook met die oog op die uitkomste van behandeling ondersoek. In vergelyking met standaard chemoterapie, het die gebruik van die TMA-tegniek met toevoeging van rituximab geen noemenswaardige verbetering in die algehele 5-jaar oorlewing (63% vs 59%, p = 0.68) óf insident-vrye oorlewing (42% vs 39%, p = 0.94) getoon nie. Insgelyks was daar geen duidelike verskille in subtipe-analise nie. Dit is egter interessant dat, met die toepassing van die Choi-kriteria, sitotoksiese middels op hul eie, in teenstelling met die geaktiveerde B-sel-subtipe, nie ‘n statisties belangrike neiging tot beter oorlewing (74% vs 55%, p = 0.32) of insident-vrye oorlewing (44% vs 40%, p = 0.42) vir die kiemsentrumsubtipe (GCB) getoon het nie. Dit was selfs nie eers moontlik om ‘n klein verskil in die teenwoordigheid van rituximab te demonstreer nie. Volgens Choi, het chemoterapie (met óf sonder die toevoeging van rituximab) by vergelyking van kiemsentrumsubtipes met nie-kiemsentrumsubtipes soortgelyke resultate gelewer as die Hans algoritme na 5-jaar oorlewing, sowel as 3-jaar insident-vrye oorlewing. Ten spyte van ’n klein steekproef het verdere subtipe-analise van BCL2- en LMO2- merkeruitdrukking van die onderskeie immunohistochemiese (IHC) subtipes die volgende aan die lig gebring: Analise met gebruik van die Choi-kriteria vir subtipe-analise vir BCL2- uitdrukking het, ongeag die subtipe (GCB of ABC) en die behandelingsmodaliteit (chemoterapie met of sonder toevoeging van rituximab), geen verskil getoon in 5-jaar oorlewing en insident-vrye oorlewing nie. Daar was egter `n noemenswaardige verskil ten opsigte van beter insident-vrye oorlewing (p = 0.0015) in die BCL2-positiewe groep van die ABC-subtipes wat met rituximab-bevattende chemoterapie behandel is. Hierdie resultate moet egter met sorg geïnterpreteer word weens die klein steekproef en kort opvolgperiode van slegs nege maande. Soortgelyke resultate met betrekking tot die uitkoms vir oorlewing is vir LMO2 gelewer, naamlik geen verskil ten opsigte van 5-jaar oorlewing of insident-vrye oorlewing ongeag die subtipe of behandelingsmodaliteit. Hier ook was daar egter beter insident-vrye oorlewing (p = 0.039) in die LMO2-positiewe groep van die ABC-subtipe wanneer rituximab-bevattende chemoterapie toegedien is. Weereens moet die resultate met sorg interpreteer word weens die klein steekproef en die kort opvolgperiode van 14 maande. BESPREKING EN GEVOLGTREKKING Die meeste studies wat reeds die verhoudings van subtipes in groot getalle DLBCL-pasiënte ondersoek het, is onder westerse populasies uitgevoer en daar bestaan onsekerheid oor of dieselfde verhoudings in Afrika van toepassing is. Terwyl 50% van alle DLBCL-pasiënte in Noord-Amerika of Europa die GCB-fenotipe uitdruk, kom dit in slegs 31% van hierdie pasiënte in Asië tot uitdrukking. Geen studie is tot dusver met behulp van die TMA-metode onder limfoomsubtipes in populasies in Afrika onderneem nie, veral nie onder pasiënte met verswakte immuunstelsels nie. Ten spyte van sekere beperkings van hierdie studie, waarvoor daar verskeie redes is, soos die verlies van analiseerbare data, die wisselende verteenwoordiging van die Suid-Afrikaanse bevolking as ‘n geheel en die klein steekproef, wat vergissing in die hand kan werk, bevestig die resultate dat limfoproliferatiewe toestande heterogeen is. Desnieteenstaande kan hierdie groep met sukses behandel word indien ‘n holistiese bestuurplan presies volgens stadiums, klassifikasie en risiko-assessering uitgevoer word – ongeag of dit in `n ontwikkelde gebied of een met min hulpbronne plaasvind. Met die beskikbaarheid van ingewikkelde metodes soos geen-ekspressie profielskepping (GEP), kan DLBCL in prognosties-voordelige kiemsentrum B-sel-agtige (GCB) en die meer aggressiewe geaktiveerde B-sel-subtipes geklassifiseer word. Teen die agtergrond van beperkte hulpbronne, is immunohistochemiese (IHC) kleuringsmetodes gebruik om - weefselmonsters wat in formalien gefikseer en in paraffien ingebed was, te analiseer. Hier was die algoritme wat oorspronklik deur Hans et al. beskryf is en deur Choi et al. verder ontwikkel is, en hoë ooreenstemming toon, vergelykbaar met die GEP-klassifikasie. Die gebruik van die IHC-gebaseerde metodologie is as ‘n eenvoudige, effektief lonende en kragtige alternatief tot GEP, wat tans as die goudstandaard vir klassifikasie van limfoom beskou word, bewys. Dit verskaf ‘n nuttige prognose-hulpmiddel vir die stratifisering van DLBCL of ander entiteite in die toekoms, selfs wanneer gevriesde weefselmonsters nie vir GEP-analise beskikbaar is nie. Onder die huidige begrotingsbeperkings in staatshospitale is middels soos rituximab by die protokolle vir die behandeling van limfoproliferatiewe toestande uitgesluit. Plaaslike terapeutiese middel-komitees beskou die nagenoeg 15% algehele oorlewingsvoordeel teen vyf jaar, wat vir DLBCL moontlik is met die toevoeging van rituximab by kombinasiechemoterapie, as te randstandig om die bykomende onkoste te regverdig. Daarvolgens is dit noodsaaklik om te demonstreer dat `n spesifieke molekulêre subtipe tot ’n beter uitkoms lei wanneer hierdie metode gebruik word en dat dit oortuigende bewyse sal lewer vir die gebruik van hierdie monoklonale teenliggaam in `n omgewing met beperkte hulpbronne.

The extracellular matrix (ECM) offers a protective shelter for cells and provides signaling paths important for cell to cell communication. ECM consists of basement membranes (BM) and interstitial matrix. BMs provide mechanical support for parenchymal cells, influence cell proliferation, survival, migration and differentiation. They are also important for tissue integrity. Laminins (LM) are the major non-collagenous component of BMs. Cell-ECM interactions, mediated by receptors, are indispensable during embryonic development, wound healing, remodeling and homeostasis of tissues. The integrins are the major cell-adhesion receptors. The expression of alpha11 integrin chain in the cornea is of great interest, as it is part of the alpha11beta1 integrin receptor for collagen type I, the predominant component of the corneal stroma. The aims were to thoroughly characterize the ECM in the developing and adult human eye, with particular focus on the cornea, LM and alpha11 integrin chains, and to examine alpha11 integrin chain in an animal model of corneal wound healing and remodeling. Human fetal eyes, 9-20 weeks of gestation (wg), and adult human corneas with different diagnosis were treated for immunohistochemistry with specific antibodies against LM and alpha11 integrin chains. Normal and knockout (ko) mice were treated with laser surgery to create a deep wound in the corneal stroma. The wound healing process was followed at different time points. The cellular source of alpha11 integrin chain was studied in cell cultures. In the fetal eyes, the BM of the corneal epithelium, the Descemet’s membrane (DM) and the Bruch’s membrane each had their specific combinations of LM chains and time line of development, whereas the lens capsule and the internal limiting membrane showed constant LM chain patterns. The epithelial BMs of normal and diseased adult corneas contained similar LM chains. The normal morphology of the epithelial BM was altered in the different diseases, particularly when scarring was present. In the scarred keratoconus corneas there were excessive LM chains. The majority of keratoconus corneas also expressed extra LM chains in the DM. At 10-17 wg alpha11 integrin chain was present in the human corneal stroma, especially in the anterior portion, but it was scarce at 20 wg, in normal adult corneas and in Fuchs’ endothelial dystrophy. In contrast, it was increased in the anterior portion of the stroma in keratoconus corneas with scarring. Alpha11 integrin ko mice had a defective healing with subsequent thinner corneas. Alpha11 integrin expression correlated to the presence of alpha-smooth muscle actin in vivo as well as in vitro. The distinct spatial and temporal patterns of distribution for alpha11 integrin and each of the LM chains suggest that they play an important role in human ocular differentiation. The selectively affected LM composition and the novel expression of alpha11 integrin chain in scarred keratoconus corneas as well as the pathologic healing in ko mice, indicate that alpha11 integrin and LM chains also play an important role in the process of corneal healing, remodeling and scarring and might participate in the pathogenesis of corneal disease. This knowledge is of practical importance for future topical therapeutic agents capable of modulating the corneal wound healing processes.

Objetivo: Avaliar in vivo a resposta tecidual de dentes de cães após perfuração de furca e recobrimento com Biodentine&trade;, em comparação ao MTA e à guta-percha, por meio de análise histopatológica, histoenzimológica, imunohistoquímica e por imunofluorescência. Métodos: Foram utilizados 30 dentes de 3 cães, divididos em 3 grupos: I - Biodentine; II - MTA; e III - Guta-Percha (controle). Após tratamento endodôntico e limpeza da câmara pulpar, perfurações no centro do assoalho foram realizadas intencionalmente em cada dente, as quais foram preenchidas com os diferentes materiais. Após 120 dias, os animais foram eutanasiados e as peças contendo os dentes e tecidos perirradiculares foram submetidas ao processamento histotécnico. Foram realizadas análises histopatológicas semi-quantitativas para avaliação da neoformação de tecido mineralizado e da reinserção de fibras, além de análise imunohistoquímica das proteínas osteopontina (OPN) e fosfatase alcalina (ALP) e imunofluorescência para proteína morfogenética óssea (BMP-2), proteína de adesão do cemento (CAP), sialoproteína óssea (BSP), osteocalcina (OCN) e proteína do cemento 1 (CEMP1) no tecido mineralizado neoformado e na região adjacente. Paralelamente, foi realizada a histoenzimologia para a atividade da TRAP e contagem dos osteoclastos. Os dados foram submetidos aos testes qui-quadrado e Kruskal-Wallis, com nível de significância de 5%. Resultados: Na avaliação do tecido mineralizado neoformado, o grupo controle foi significantemente diferente dos demais grupos (p<0,0001), sendo que não houve formação de tecido mineralizado em nenhum espécime desse grupo. Nos grupos tratados com MTA e Biodentine houve formação de tecido mineralizado em 88% e 92% dos espécimes, respectivamente, sem diferença entre eles (p>0,05). Ainda, o grupo controle apresentou fibras colágenas paralelas à perfuração. Nos grupos tratados com MTA ou Biodentine também houve fibras colágenas paralelas à perfuração, porém com algumas fibras reinseridas perpendicularmente em diferentes áreas do tecido mineralizado neoformado. Todos os tratamentos induziram a expressão de OPN e ALP, porém em menor intensidade no grupo controle e em maior intensidade no grupo tratado com MTA (p<0,05). Apenas o tecido mineralizado formado após o tratamento com MTA expressou BMP-2, BSP, OCN, CAP e CEMP1. Com relação à avaliação dos osteoclastos, não foi possível encontrar diferença estatística entre os grupos (p=0,97). Conclusão: Com base nos parâmetros analisados, pôde-se concluir que o MTA e a Biodentine apresentaram resposta tecidual satisfatória, com formação de tecido mineralizado e reinserção parcial de fibras, podendo ser indicados para o selamento de perfurações de furca. Além disso, o presente estudo elucidou alguns mecanismos de ação pelo quais o MTA e a Biodentine induzem a formação do tecido mineralizado, com expressão dos marcadores da mineralização ALP e OPN, sem interferência na quantidade de osteoclastos. Apenas o MTA estimulou a expressão de proteínas associadas à formação de tecido mineralizado semelhante ao cemento / Aim: This study evaluated in vivo tissue response in dogs teeth after sealing of furcation perforations with Biodentine&trade;, MTA and gutta-percha, by means of histopathological, histoenzymological, immunohistochemical and immunofluorescence analysis. Methods: Thirty teeth of 3 dogs were used, divided in 3 groups: I - Biodentine; II - MTA; and III - Guta-Percha (control). After endodontic treatment, perforations were made on the pulp chamber floor and filled with the different materials. The animals were euthanized after 120 days and the teeth and surrounding tissues were processed for histopathological analysis of new mineralized tissue formation and collagen fiber reinsertion, immunohistochemical analysis of osteopontin (OPN) and alkaline phosphatase (ALP) and immunofluorescence analysis for bone morphogenetic protein (BMP-2), cementum attachment protein (CAP), bone sialoprotein (BSP), osteocalcin (OCN) and cementum protein1 (CEMP1). Histoenzymology was performed for TRAP activity and osteoclast count. Data were submitted to chi-square and Kruskal-Wallis tests (=0.05). Results: Gutta-percha did not induce mineralized tissue formation. MTA and Biodentine formed mineralized tissue in 88% and 92% of specimens, respectively, with no significant difference (p>0.05). In addition, the control group had collagen fibers parallel to the perforation. In the groups treated with MTA or Biodentine there were also collagen fibers parallel to the perforation, but with some fibers reinserted perpendicularly in different areas of the neoformed mineralized tissue. All materials induced OPN and ALP expression, weakest for gutta-percha and strongest for MTA (p<0.05). Only MTA induced BMP-2, BSP, OCN, CAP and CEMP1 expression. Osteoclast count was similar in the groups (p=0.97). Conclusion: Thus, according to the parameters analyzed in this present study, MTA and Biodentine presented satisfactory tissue response, with formation of mineralized tissue and partial reinsertion of fibers, and could be indicated for sealing furcation perforations. In addition, the present study elucidated some mechanisms of action by which MTA and Biodentine induce the formation of mineralized tissue, with expression of ALP and OPN mineralization markers, without interference in number of osteoclasts. Only MTA stimulated the expression of proteins associated with the formation of a cement-like mineralized tissue

O objetivo desse estudo in vivo foi avaliar a intensidade de expressão de moléculas indicadoras da diferenciação celular com fenótipo mineralizador em dentes de cães com rizogênese incompleta e lesão periapical induzida experimentalmente submetidos à irrigação dos canais radiculares por pressão apical negativa (EndoVac®) e à irrigação por pressão apical positiva (irrigação convencional). Foram utilizados 30 dentes (60 raízes), divididos em 3 grupos: Grupo EndoVac® Irrigação com pressão apical negativa (n=20), Grupo Convencional Irrigação com pressão apical positiva (n=20); e Grupo Controle com Lesão Periapical (n=20). Após indução de lesões periapicais, os canais radiculares dos grupos Endovac e Convencional foram instrumentados com limas manuais, empregando o sistema de irrigação correspondente. Decorridos 90 dias, os animais foram eutanasiados e os espécimes submetidos ao processamento histotécnico. Cortes representativos de cada grupo foram corados com hematoxilina e eosina (HE), para descrição histopatológica das regiões apical e periapical. Os cortes foram submetidos à para marcação de osteopontina (OPN), fosfatase alcalina (ALP) e fator de transcrição RUNX2 nas regiões apical e periapical das raízes. Para avaliação da intensidade da expressão desses marcadores foi realizada uma análise semi-quantitativa por meio de um sistema de escores. Os dados foram analisados estatisticamente pelo teste não-paramétrico de Kruskal-Wallis e pós-teste de Dunn e o nível de significância adotado foi de 5%. Com relação à descrição histopatológica, achados distintos foram observados em cada grupo. Resumidamente, o grupo Endovac demonstrou um tecido conjuntivo bem organizado, invaginando-se para o interior do canal radicular, e exibiu um processo de reparo mais avançado, com relação aos demais grupos. A avaliação das imunomarcações para RUNX2 revelou que no grupo Endovac® houve marcação significantemente mais intensa (p=0,03), em comparação ao grupo controle. Com relação à expressão de OPN, não foi possível encontrar diferença estatisticamente significante entre os grupos (p=0,15). Após análise das imunomarcações para ALP, observou-se diferença estatisticamente significante entre os grupos (p=0,0021), sendo que o grupo Endovac® apresentou marcação significantemente mais intensa com relação ao grupo controle. Os resultados do presente estudo in vivo permitiram concluir que a irrigação por pressão apical negativa (EndoVac®) apresentou potencial mineralizador, indicando ser o melhor sistema de irrigação para dentes com ápice incompleto e lesão periapical / The objective of this in vivo study was to evaluate the expression intensity of molecules indicating cell differentiation with mineralizing phenotype in immature dogs teeth with experimentally induced apical periodontitis subjected to irrigation of root canals using negative apical pressure (EndoVac®) and positive apical pressure (conventional irrigation). Thirty teeth (60 roots) were divided into 3 groups: EndoVac® Group - negative apical pressure irrigation (n=20), Conventional Group - positive apical pressure irrigation (n=20); and Control Group with apical periodontitis (n=20). After induction of periapical lesions, the root canals of the EndoVac ® and Conventional groups were instrumented with hand files, using the corresponding irrigation system. After 90 days, the animals were euthanized and the specimens were submitted to histotechnical processing. Representative sections of each group were stained with hematoxylin and eosin (HE) for histopathological description of the apical and periapical regions. The sections were submitted to osteopontin (OPN), alkaline phosphatase (ALP) and RUNX2 transcription factor Immunostaining in the apical and periapical regions of the roots. A semi-quantitative analysis was performed to evaluate the expression intensity of these markers, using a score system. Data were statistically analyzed by the Kruskal-Wallis non-parametric test and Dunn post-test, and the significance level was set at 5%. Regarding the histopathological description, different findings were observed in each group. Briefly, the EndoVac® group demonstrated a well-organized connective tissue, invaginating into the root canal, and exhibited a more advanced repair process in relation to the other groups. RUNX2 immunostaining revealed that in the EndoVac® group there was a significantly stronger (p=0.03) immunostaining in comparison to the control group. Regarding the OPN expression, it was not possible to find a statistically significant difference between the groups (p=0.15). After analyzing ALP immunostaining, a statistically significant difference was observed between the groups (p=0.0021), and the EndoVac® group showed a markedly stronger mark immunostaining than the control group. The results of the present in vivo study allowed concluding that negative apical pressure irrigation (EndoVac®) presented mineralizing potential, indicating that it is the best irrigation system for teeth with an open apex and apical periodontitis

As lesões periapicais ocasionam destruição dos tecidos apicais e periapicais, por meio da exacerbação da resposta inflamatória e imune. O sistema imunológico é ativado e células são recrutadas para o local da lesão, incluindo os macrófagos, que podem ser polarizados em macrófagos M1 e M2.O presente estudo teve como objetivo quantificar macrófagos M1 e M2 em cistos periapicais de dentes decíduos e permanentes.Foram selecionados 15 casos de cistos periapicais de dentes decíduos e 10 cistos periapicais de dentes permanentes. Em todos os casos foi realizada análise histopatológica em HE, classificando o tipo e o grau do infiltrado inflamatório, por meio de escores. Além disso, foi realizada a quantificação dos marcadores CD68 (M1+, M2+) e CD163 (M1-,M2+) por meio da análise imunohistoquímica. Os resultados obtidos foram submetidos ao teste de Mann Whitney, com nível de significância de 5%.Embora tenham sido detectados macrófagos M1 e M2 tanto nas lesões dos dentes decíduos quanto dos permanentes, houve maior prevalência de macrófagos M2 (p<0,05). A comparação entre dentes decíduos e permanentes evidenciou maior quantificação de macrófagos M1 nas lesões dos dentes permanentes (p=0,002). Pôde-se concluir que os macrófagos M1 e M2 estão presentes nos cistos periapicais de dentes decíduos e permanentes, com maior quantidade de células M2. Além disso, os cistos periapicais dos dentes permanentes apresentaram maior quantidade de macrófagos M1, em comparação aos cistos dos dentes decíduos / The radicular lesion leads to the apical and periapical tissues destruction throughout an inflammatory and immune response. The immune system is activated and cells are recruited to the lesion site, which includes macrophages that can be polarized into M1 and M2 macrophages. The objective of this study was to quantify M1 and M2 macrophages in radicular cysts in permanent and deciduous teeth. In total, 15 radicular cysts cases in deciduous teeth and 10 in permanent teeth were selected. A histopathologic analysis in HE was performed, allowing the type and inflammatory infiltrates level classification in scores. In addition, the CD68 (M1+, M2+) and CD163 (M1-, M2+) markers were quantified through an immunohistochemistry analysis. The data acquired were submitted to a Mann Whitney test, with a 5% significance level. A higher prevalence of M2 macrophages (p<0.05) was observed, despite both M1 and M2 macrophages have been detected in the permanent and deciduous teeth lesions. The comparison between permanent and deciduous teeth presented a higher M1 macrophages quantity in permanent teeth lesions (p=0.002). In summary, the M1 and M2 macrophages are present in deciduous and permanent teeth radicular cysts, with a higher quantity of M2 cells. Moreover, the radicular cysts in permanent teeth have shown a higher M1 macrophages quantity when compared to cysts in deciduous teeth