Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

Linfomas pertencem a um grupo de neoplasias em que há proliferação monoclonal de linfócitos malignos, sendo uma das neoplasias mais frequentemente diagnosticadas em cães. Podem ser classificados quanto à forma anatômica em multicêntrico, mediastinal, digestório e extranodal. Dentre os extranodais, os linfomas cutâneos são classificados histologicamente como epiteliotrópicos e não epiteliotrópicos e são predominantemente de imunfenótipo T, com raros casos do tipo B. A principal característica histopatológica do linfoma epiteliotrópico em cães é o tropismo das células neoplásicas pela epiderme, mucosa ou estruturas anexas, enquanto o linfoma não epiteliotrópico é caracterizado pela infiltração dérmica e subcutânea sem invasão das estruturas anexas. Os linfomas cutâneos caninos têm progressão rápida, são considerados bastante agressivos e com mau prognóstico, com baixa taxa de resposta à quimioterapia. Um dos fatores que podem contribuir para isso é a resistência das células a múltiplas drogas e entre esses mecanismos de resistência estão o efluxo de drogas do meio intracelular para o extracelular por meio dos transportadores da família ABC, como a glicoproteína-P, MRP (multiple resistance protein) e BCRP (breast câncer resistance protein) e da LRP (lung resistance protein), uma proteína vault responsável pelo transporte nucleocitoplasmático. O objetivo deste estudo foi caracterizar imunofenotipicamente os linfomas cutâneos, a proliferação celular por meio do marcador Ki67, a expressão das proteínas de resistência glicoproteína-P, MRP, BCRP e LRP e avaliar a relação dessas proteínas com a sobrevida dos animais. Foi realizado um estudo retrospectivo com 21 casos de cães linfomas cutâneos com diagnóstico histopatológico. A técnica de imunoistoquímica foi utilizada para determinar a imunofenotipagem dos linfomas pelos marcadores CD3 e CD20, a proliferação celular por Ki67 e a expressão de glicoproteína-P, MRP, BCRP e LRP. Dos 21 animais, 38% tiveram diagnóstico histopatológico de linfoma epiteliotrópico, 52% eram linfomas não epiteliotrópicos, 5% dos casos de linfoma não tiveram epiteliotropismo definido e 5% foram classificados como neoplasia de células redondas. O imunofenótipo predominante foi CD3+CD20- (76%), 15% dos casos eram CD3-CD20+ e 9% eram CD3+CD20+. A mediana de células marcadas para Ki67 foi de 31%. Com relação aos marcadores de resistência a múltiplas drogas, a mediana da marcação de glicoproteína-P foi de 40%, a de LRP foi de 65% enquanto para MRP e BCRP, 19% e 23%, respectivamente. Os linfomas cutâneos não epiteliotrópicos foram mais frequentes que os epiteliotrópicos e o imunfenótipo predominante foi o T. A ocorrência de linfócitos CD3-CD20+ e CD3+CD20+ indica a necessidade de mais estudos e um painel mais amplo de anticorpos para subtipagem desses linfomas. A glicoproteína-P teve maior expressão nos linfomas não epiteliotrópicos do que nos epiteliotrópicos e não houve correlação entre as proteínas de resistência e o tempo de sobrevida dos animais, sugerindo que, além da biologia da neoplasia, outros mecanismos de resistência a múltiplas drogas diferente dos estudados possam ter um papel relevante na baixa resposta do linfoma cutâneo à quimioterapia. / Lymphoma is a group of blood cell tumors that develop from monoclonal proliferation of malignant lymphocytes. Lymphoma is the most frequent neoplasia in dogs and can be anatomically classified in multicentric, mediastinal, digestive and extranodal. Cutaneous lymphomas an extranodal type of lymphoma are classified histologically in epitheliotropic and non-epitheliotropic and are predominantly of T-cell immunophenotype, and rare cases of B cell phenotype. The main histopathological characteristic of epitheliotropic lymphoma in dogs is the tropism of neoplastic cells by the epidermis, mucosa or adjacent structures, while non-epitheliotropic lymphoma is characterized by dermal and subcutaneous infiltration without invasion of adjacent structures. Canine cutaneous lymphomas have rapid progression, are considered very aggressive and have poor prognosis. These dogs, usually have a low rate of response to chemotherapy which can be associated to an antineoplastic resistance. Among mechanisms of resistance are efflux of drugs from intracellular to extracellular through ABC family transporters such as P-glycoprotein, MRP (multple resistance protein) and BCRP (breast cancer resistance protein) and LRP (lung resistance protein), a vault protein responsible for nucleocytoplasmic transport. The aim of this study was to characterize immunophenotypically cutaneous lymphomas, measure cell proliferation using the Ki67 marker, the expression of resistance proteins P-glycoprotein, MRP, BCRP and LRP and to evaluate the relationship of these proteins with the survival of the animals. A retrospective study was performed with 21 cases of dogs with cutaneous lymphoma with histopathological diagnosis. Immunohistochemical was used to immunophenotyping of lymphomas by CD3 and CD20 markers, Ki67 cell proliferation, and P-glycoprotein, MRP, BCRP and LRP expression. Of the 21 animals, 38% had histopathological diagnosis of epitheliotropic lymphoma, 52% were non-epitheliotropic lymphomas, 5% of lymphoma cases had no definition and 5% were classified as round cell neoplasia. The predominant immunophenotype was CD3+CD20- (76%), 15% of the cases were CD3-CD20 + and 9% were CD3 + CD20 +. The median of cells labeled for Ki67 was 31%. Regarding the markers of resistance to multiple drugs, the median of the P-glycoprotein label was 40%, which 65% of LRP while for MRP and BCRP, 19% and 23%, respectively. Non-epitheliotropic cutaneous lymphomas were more frequent than epitheliotropic lymphomas and the predominant immunophenotype was T. The occurrence of CD3-CD20+ and CD3+CD20+ lymphocytes indicates the need for further studies and a wider panel of antibodies for subtyping these lymphomas. P-glycoprotein had higher expression in non-epitheliotropic lymphomas than in epitheliotropic lymphomas and there was no correlation between resistance proteins and survival time of the animals, suggesting that in addition to the biology of neoplasia other mechanisms of resistance to multiple drugs different from those studied may play a relevant role in the low response of cutaneous lymphoma to chemotherapy.

Linfomas pertencem a um grupo de neoplasias em que há proliferação monoclonal de linfócitos malignos, sendo uma das neoplasias mais frequentemente diagnosticadas em cães. Podem ser classificados quanto à forma anatômica em multicêntrico, mediastinal, digestório e extranodal. Dentre os extranodais, os linfomas cutâneos são classificados histologicamente como epiteliotrópicos e não epiteliotrópicos e são predominantemente de imunfenótipo T, com raros casos do tipo B. A principal característica histopatológica do linfoma epiteliotrópico em cães é o tropismo das células neoplásicas pela epiderme, mucosa ou estruturas anexas, enquanto o linfoma não epiteliotrópico é caracterizado pela infiltração dérmica e subcutânea sem invasão das estruturas anexas. Os linfomas cutâneos caninos têm progressão rápida, são considerados bastante agressivos e com mau prognóstico, com baixa taxa de resposta à quimioterapia. Um dos fatores que podem contribuir para isso é a resistência das células a múltiplas drogas e entre esses mecanismos de resistência estão o efluxo de drogas do meio intracelular para o extracelular por meio dos transportadores da família ABC, como a glicoproteína-P, MRP (multiple resistance protein) e BCRP (breast câncer resistance protein) e da LRP (lung resistance protein), uma proteína vault responsável pelo transporte nucleocitoplasmático. O objetivo deste estudo foi caracterizar imunofenotipicamente os linfomas cutâneos, a proliferação celular por meio do marcador Ki67, a expressão das proteínas de resistência glicoproteína-P, MRP, BCRP e LRP e avaliar a relação dessas proteínas com a sobrevida dos animais. Foi realizado um estudo retrospectivo com 21 casos de cães linfomas cutâneos com diagnóstico histopatológico. A técnica de imunoistoquímica foi utilizada para determinar a imunofenotipagem dos linfomas pelos marcadores CD3 e CD20, a proliferação celular por Ki67 e a expressão de glicoproteína-P, MRP, BCRP e LRP. Dos 21 animais, 38% tiveram diagnóstico histopatológico de linfoma epiteliotrópico, 52% eram linfomas não epiteliotrópicos, 5% dos casos de linfoma não tiveram epiteliotropismo definido e 5% foram classificados como neoplasia de células redondas. O imunofenótipo predominante foi CD3+CD20- (76%), 15% dos casos eram CD3-CD20+ e 9% eram CD3+CD20+. A mediana de células marcadas para Ki67 foi de 31%. Com relação aos marcadores de resistência a múltiplas drogas, a mediana da marcação de glicoproteína-P foi de 40%, a de LRP foi de 65% enquanto para MRP e BCRP, 19% e 23%, respectivamente. Os linfomas cutâneos não epiteliotrópicos foram mais frequentes que os epiteliotrópicos e o imunfenótipo predominante foi o T. A ocorrência de linfócitos CD3-CD20+ e CD3+CD20+ indica a necessidade de mais estudos e um painel mais amplo de anticorpos para subtipagem desses linfomas. A glicoproteína-P teve maior expressão nos linfomas não epiteliotrópicos do que nos epiteliotrópicos e não houve correlação entre as proteínas de resistência e o tempo de sobrevida dos animais, sugerindo que, além da biologia da neoplasia, outros mecanismos de resistência a múltiplas drogas diferente dos estudados possam ter um papel relevante na baixa resposta do linfoma cutâneo à quimioterapia. / Lymphoma is a group of blood cell tumors that develop from monoclonal proliferation of malignant lymphocytes. Lymphoma is the most frequent neoplasia in dogs and can be anatomically classified in multicentric, mediastinal, digestive and extranodal. Cutaneous lymphomas an extranodal type of lymphoma are classified histologically in epitheliotropic and non-epitheliotropic and are predominantly of T-cell immunophenotype, and rare cases of B cell phenotype. The main histopathological characteristic of epitheliotropic lymphoma in dogs is the tropism of neoplastic cells by the epidermis, mucosa or adjacent structures, while non-epitheliotropic lymphoma is characterized by dermal and subcutaneous infiltration without invasion of adjacent structures. Canine cutaneous lymphomas have rapid progression, are considered very aggressive and have poor prognosis. These dogs, usually have a low rate of response to chemotherapy which can be associated to an antineoplastic resistance. Among mechanisms of resistance are efflux of drugs from intracellular to extracellular through ABC family transporters such as P-glycoprotein, MRP (multple resistance protein) and BCRP (breast cancer resistance protein) and LRP (lung resistance protein), a vault protein responsible for nucleocytoplasmic transport. The aim of this study was to characterize immunophenotypically cutaneous lymphomas, measure cell proliferation using the Ki67 marker, the expression of resistance proteins P-glycoprotein, MRP, BCRP and LRP and to evaluate the relationship of these proteins with the survival of the animals. A retrospective study was performed with 21 cases of dogs with cutaneous lymphoma with histopathological diagnosis. Immunohistochemical was used to immunophenotyping of lymphomas by CD3 and CD20 markers, Ki67 cell proliferation, and P-glycoprotein, MRP, BCRP and LRP expression. Of the 21 animals, 38% had histopathological diagnosis of epitheliotropic lymphoma, 52% were non-epitheliotropic lymphomas, 5% of lymphoma cases had no definition and 5% were classified as round cell neoplasia. The predominant immunophenotype was CD3+CD20- (76%), 15% of the cases were CD3-CD20 + and 9% were CD3 + CD20 +. The median of cells labeled for Ki67 was 31%. Regarding the markers of resistance to multiple drugs, the median of the P-glycoprotein label was 40%, which 65% of LRP while for MRP and BCRP, 19% and 23%, respectively. Non-epitheliotropic cutaneous lymphomas were more frequent than epitheliotropic lymphomas and the predominant immunophenotype was T. The occurrence of CD3-CD20+ and CD3+CD20+ lymphocytes indicates the need for further studies and a wider panel of antibodies for subtyping these lymphomas. P-glycoprotein had higher expression in non-epitheliotropic lymphomas than in epitheliotropic lymphomas and there was no correlation between resistance proteins and survival time of the animals, suggesting that in addition to the biology of neoplasia other mechanisms of resistance to multiple drugs different from those studied may play a relevant role in the low response of cutaneous lymphoma to chemotherapy.

Zunächst wurden mit Hilfe eines neuen Injektionssystems die Vorteile der Bolusgabe und der kontinuierlichen Applikation d-galaktosehaltiger Ultraschallsignalverstärker kombiniert und an einem klein- (Æ 0,86mm) und einem großlumigen (Æ 16 mm) Gefäßmodell die optimale Basis- und Bolusflußratenkombination für die Powerdopplersonographie evaluiert. Der Powerdopplersignalintensitätsverlauf verschiedener Infusionsprotokolle wurde subjektiv von drei Beobachtern einer sechsstufigen Skala zugeordnet. Objektive Intensitätsmeßdaten wurden mit einer CW-Dopplersonde und einem Computersystem gewonnen. Die 1 ml/min.-Basisinfusionsrate kam dem Optimalzustand am nächsten (Großlumenmodell: 70 %, Kleinlumenmodell: 100 % Grad-4-Plateau trotz geringer Standardabweichung der mittleren Spektraldopplerverstärkungsmeßwerte im Plateaubereich). Sie ermöglichte eine annähernd optimale Gefäßdarstellung mit ausreichendem Spielraum für mehrfache, optisch gut wahrnehmbare Bolusgaben (Optimalbolus: 300mg/ml, 2 ml/s, 1 s Injektionsdauer). Diese Ergebnisse lassen eine medizinische und ökonomische Optimierung des Signalverstärkereinsatzes in der Powerdopplersonographie erwarten. Anhand von tierexperimentellen Untersuchungen wurde die Zuverlässigkeit der nativen und der signalverstärkten Farbdopplersonographie bei der Visualisierung der intratumorösen Angioneogenese im Vergleich zur histologischen Vaskularisationsquantifizierung evaluiert. Zu diesem Zweck wurden Melanomzellen des Typs B16-F1 Mäusen intra- bzw. subkutan imjiziert und nach einer Wachstumsperiode in 54 Fällen sonographisch im B-Modus bezüglich Größe, Homogenität, Echogenität und Grenzechoschärfe und in der Farbdopplersonographie bezüglich intratumorös erkennbarer Gefäßzahl, Gefäßarchtektur und Gefäßanteil an der Tumorquerschnittsfläche (= percentage vessel area, PVA) analysiert. Die anschließende histologische Aufarbeitung erbrachte Vergleichsdaten hinsichtlich Größe, Nekrotisierung und Vaskularisation der Tumoren. Es fand sich eine signifikante Korrelation (p < 0,01) zwischen den makroskopisch und den sonographisch ermittelten Tumordurchmessern, jedoch zwischen Histologie und Sonographie bezüglich sonographisch erkannter Nekrosen nur, wenn diese mehr als 40 % des Tumors erfaßten. Nach Signalverstärkerapplikation stiegen die sonographisch erkennbare Tumorgefäßzahl, -architektur und die PVA signifikant an (p < 0,01), letztere bei den intrakutanen Tumoren um 483 % und bei den subkutanen um 373 %. Der histologische Vaskularisationsgrad korrelierte am stärksten (r = 0,686) von allen Farbdopplerparametern mit der signalverstärkten PVA (p < 0,01). Somit trug die Signalverstärkergabe zur Verbesserung der Korrelation der Ergebnisse der sonographischen und histologischen Vaskularisations- und Angioneogenesebeurteilung bei. Im klinisch-experimentellen Teil wurde prospektiv bei 83 malignitätssuspekten, kutan oder subkutan lokalisierten Raumforderungen unterschiedlichen, meist epithelialen Ursprungsgewebes mittels nativer und signalverstärkter Farbduplexsonographie eine Dignitätseinschätzung vorgenommen. Als Einstufungskriterien wurden diverse B-Modus- und Farbdopplerkriterien, u.a. intratumorös erkennbare Gefäßzahl und PVA, verwendet. Die Diagnosesicherung erfolgte durch Histologie oder Verlaufskontrolle. Während weder die B-Modus-Kriterien noch die Dopplerspektralanalyse für die Dignitätseinschätzung geeignet erschienen, trugen die Farbdopplerparameter und besonders die PVA nach Signalverstärkergabe zur Dignitätsbestimmung bei, wobei die erkennbare Gefäßzahl nach Signalverstärkergabe signifikant (p < 0,01) zunahm. Als ungeeignet erwiesen sich diese Kriterien jedoch bei entzündlichen und angiomatösen Raumforderungen und epithelialen Rezidivtumoren. Insgesamt liefert die signalverstärkte Farbdopplersonographie wesentliche zusätzliche Informationen bezüglich Angioneogenese und Dignität von Raumforderungen der Haut und ihrer Anhangsgebilde, insbesondere die signalverstärkte semiquantitative Vaskularisationsanalyse. Sie ist der B-Modus-Sonographie und der Spektraldoppleranalyse überlegen. Die Signalverstärkergabe führt zu einer deutlichen Sensitivitätserhöhung. / A new injection system combines the advantages of bolus and of continuous application of ultrasound contrast agents. This study evaluated the optimal combination of basic and bolus flow rates in power Doppler sonography using two different types of vitro vessel models. The flow of an in vitro circulation model consisting of a wide or small lumen vessels was visualized by power Doppler ultrasound. The intensity of colour Doppler signal was assessed subjectively by three observers using a six step scale (optimal: degree 5). The objective signal intensity was measured by a CW-Doppler probe and a computed sytem. Using a basic flow rate of 1 ml/min., approximately optimal results were reached. This basic flow rate enabled an almost optimal visualization of the vessel lumen with sufficient free range for repeated, well visible bolus injections. Thus, the use of the new injection system combining bolus and continous injection of contrast agent improves medical and economic use of signal enhancing agents in power Doppler sonography. The aim of the next study was to evaluate the reliability of plain and enhanced colour Doppler sonography in visualization of intratumoral vascularization as a sign of malignant angioneogenesis. Therefore, malignant melanomas of the histological type B16-F1 which had been implanted in mice were examined by sonography. The majority of these tumours was localized intracutaneously, the minority subcutaneously. Various B-mode aspects, colour Doppler criteria, and spectral Doppler parameter were evaluated before and after i.v.-application of an ultrasound signal enhancer. After sonographic examination, all tumors were analyzed histologically with semiquantitative grading of tumoral vascularization. Despite the higher mean volume of the subcutaneous tumours, the percentage of tumors with visible intratumoral vessels was not higher than in intracutaneous tumors on plain images. Enhanced, the sonographical vascularization of subcutaneous tumors seemed to be superior to this of the intracutaneous. The signal enhanced mode was definitely superior to plain Doppler in showing the intratumoral vessels as a sign of angioneogenesis. The intratumoral vascular structure could be sufficiently analyzed the minority of all tumors by plain Doppler, but in more than 90 % after application of the signal enhancing agent. Intracutaneous tumors had a more compact vascular structure than subcutaneous. Despite the missing direct correlation between the sonographically and the histologically determined degree of tumour vascularization the correlation was improved after application of the signal enhancer. Only the enhanced colour Doppler sonography provides valuable noninvasive information about angioneogenetic hypervascularization in experimental melanomas. The third study evaluated colour Doppler criteria to differentiate between malignant and benign skin tumors determining the extent of the intratumorous vascularization. The B-mode sonomorphology and the extent of the vascularization in color Doppler of clinically potentially malignant tumours of the cutaneous and subcutaneous structures were analyzed and quantified by different methods. After application of the signal enhancing agent, counting the intratumoral vessels visible on one ultrasound slice led to the highest sensitivity of all used criteria but the specificity was poor. The highest specificity and diagnostic accuracy of all used criteria were obtained using the criterion "percentage vessel area > 5,0 %" after excluding all inflamed lesions by clinical aspects. Plain colour Doppler increased the specificity but the sensitivity decreased to a not acceptable level. The analysis of the B-mode morphology and spectral Doppler parameter of intratumoral vessels did not contribute to differential diagnosis. Signal enhanced colour Doppler sonography is a valuable tool in pretherapeutic assessment of cutaneous lesions. This method can be relevant for therapy and prognosis. The application of the signal enhancing agent increases significantly (p < 0.01) the sensitivity. This method is not useful in assessing benignity or malignancy of inflamed lesions and angiomas.