Evaluation of the immunomodulatory activity of thalidomide on tumor-associated macrophages in the 4T1 murine metastatic breast cancer model / [Avaliação do efeito imunomodulador da talidomida em macrófagos associados a tumores no modelo murino de câncer de mama metastático 4T1]

The present work evaluated the immunomodulatory effect of thalidomide (Thal) at different doses on tumor-associated macrophages (TAMs) using a mouse model of human breast cancer. Mice were inoculated with 4T1 cells in the left flank and treated with Thal once a day at concentrations of 50, 100, and 150mg/kg body weight from the 5th day until the 28th day of tumor inoculation. The tumors were sized, proliferation index and TAMs count were evaluated in primary tumors and metastatic lungs. In addition, the metastasis rate was evaluated in the lungs. Thal at 150mg/kg significantly decreased tumor growth, proliferation index, and TAMs infiltration in primary tumors. Conversely, a higher number of TAMs and lower proliferation index were observed in metastatic lungs in mice treated with 150mg/kg of Thal. Furthermore, Thal at 150mg/kg significantly decreased the metastatic nodules in the lungs. Our findings demonstrated that Thal treatment considerably decreased the primary tumor and lung metastasis in mice associated with different TAM infiltration effects in these sites.(AU)

No presente trabalho, foi avaliado o efeito imunomodulador de diferentes doses de talidomida em macrófagos associados ao tumor (TAMs), em um modelo murino de câncer de mama. Camundongos foram inoculados com células 4T1, na região do flanco esquerdo, e tratados com talidomida, uma vez ao dia, nas doses de 50, 100 e 150mg/k, por massa corporal, do quinto dia ao 28º dia de inoculação tumoral. Os tumores foram medidos, o índice de proliferação celular e a contagem de TAMs foram avaliados nos tumores primários e nos pulmões com metástases. Além disso, a taxa de metástases pulmonares também foi avaliada. A talidomida na dose de 150mg/kg diminuiu significativamente o crescimento tumoral, o índice de proliferação celular e a infiltração de TAMs nos tumores primários. Por outro lado, maior número de TAMs e menor índice de proliferação celular foram observados nos pulmões metastáticos, em camundongos tratados com 150mg/kg de talidomida. Ademais, a talidomida na dose de 150mg/kg diminuiu significativamente os nódulos metastáticos nos pulmões. Os resultados demonstraram que o tratamento com talidomida diminuiu o crescimento tumoral e as metástases pulmonares em camundongos, associado com diferentes efeitos na infiltração de TAMs nesses locais.(AU)

Angiopreventive versus angiopromoting effects of allopurinol in the murine sponge model.

Recent data has indicated that, besides its classical therapeutic indication in hyperurecemia and gout, xanthine oxidase inhibitors can be used to various forms of ischemia and other types of tissue and vascular injuries. We tested the hypothesis that allopurinol, an inhibitor of xanthine oxidase (XO), might modulate acute and/or chronic inflammatory angiogenesis induced by subcutaneous implantation of synthetic matrix in mice. C57/BL6 male mice (6-7 weeks) were implanted with polyether-polyurethane sponge discs. The animals received by oral gavage 1.0mg/kg of allopurinol for six consecutive days in two treatment regimen. In the first series of experiments, the treatment was initiated 24h post-implantation and the implants were removed at day 7 post-implantation. For the assessment of the effect of the compound on chronic inflammation, the treatment was initiated at day 8 post-implantation and the implants removed 14days post-implantation. Angiogenesis as determined by hemoglobin content, VEGF levels and number of vessels intraimplant, and inflammation (myeloperoxidase -MPO, n-acetyl-ß-d-glucosaminidase -NAG, TNF-α and CCL2 levels) were reduced by allopurinol treatment in acute phase. Similarly, the treatment inhibited nitric oxide and H2O2 production. However, fibrogenesis determined by collagen deposition and levels of TGF-ß1 increased in the implants after allopurinol treatment. In marked contrast with the effects when the treatment initiated 24h post-implantation, allopurinol increased angiogenesis and inflammation but reduced collagen and TGF-ß1 levels intra-implant, when the treatment was started during the chronic inflammatory process. The dual effects of allopurinol described here, extend its range of actions as a potential agent able to modulate the components of the fibrovascular tissue present in both physiological (healing processes) as well as in chronic fibroproliferative diseases. These modulatory effects depended on the phase at which the treatment was initiated.

Distinct types of tumors exhibit differential grade of inflammation and angiogenesis in mice.

Inflammation, angiogenesis and cytokine production are common features of almost, if not all tumors. However, the extent of these processes induced by different types of tumors has not been evaluated. We investigated the growth pattern of the experimental metastatic tumors, B16F10 melanoma, CT26.WT colon and 4T1 mammary cells inoculated in the flank of syngeneic mice and determined the degree of inflammation, angiogenesis, and production level of pro-inflammatory and pro-angiogenic cytokines within the tumors. In addition, we have analyzed vascular changes in the interface between the tumors and the adjacent cutaneous tissue and levels of relevant pro-inflammatory and pro-angiogenic cytokines systemically. The weight of tumors 15 days post-inoculation of 10(6) cells was markedly different. Melanomas were 2 and 10-fold heavier than colon and mammary tumors, respectively. Locally, CT26.WT tumor cells induced more vessels in cutaneous tissue adjacent to the tumors but systemically, the plasma levels of VEGF were higher (approximately 2-fold) in 4T1 tumor-bearing mice compared with the other two tumors. Mammary tumors presented the most prominent inflammatory content as assessed by a range of markers (inflammatory enzymes and cytokines). The vascular index, as determined by the intra-tumor amount of hemoglobin and number of vessels in hot spot areas, was also higher (approximately 2-fold) in melanomas compared with the other two tumors. These findings showing that distinct tumor types determine differential grade of inflammation, angiogenesis and host interaction in mice may provide new insights to tailor differential therapeutic approach based on the status of tumor biomarkers.

Marcadores de proliferação celular no tumor venéreo transmissível canino transplantado / Cell proliferation markers in the transplanted canine transmissible venereal tumor

Adult male mongrel dogs were subcutaneously transplanted with the canine transmissible venereal tumor (TVT) on the hypogastric region. Twelve specimens of tumors were collected, half during the proliferative phase and the other half during the regressive phase. Fragments of the tumor were fixed in 10 percent buffered formalin and routinely processed for light microscopy. Sections of 4µm were stained by Schorr or AgNOR or either immunostained for MIB1 (Ki67). Schorr stain, AgNOR and MIB1 showed an increased proliferative activity through mitotic index, nuclear argyrophilic protein stain and cycling tumoral cells in the growing tumors, respectively. All of the three cell proliferation markers were able to distinguish the TVT in both evolution phases. MIB1 monoclonal antibody was the best in the morphologic evaluation of growth and regression of TVT. This resulted in higher values than AgNORs counting and mitotic index. MIB1 immunostaining was the most effective parameter of the proliferative activity of TVT. However, a significant correlation has been detected only between mitosis counting and AgNORs.(AU)

Cães machos, adultos, mestiços, foram transplantados com células do tumor venéreo transmissível canino (TVTC), na região hipogástrica. Foram coletados doze espécimes do TVTC, sendo metade durante a fase proliferativa e metade durante a fase regressiva. Fragmentos do tumor foram fixados em formol a 10 por cento, tamponado e processado rotineiramente para microscopia de luz. Secções de 4µm foram coradas pelo Shorr, ou pela AgNOR, ou ainda, imunocorado para MIB 1 (Ki67). As colorações pelo Shorr, AgNOR, ou MIB 1 mostraram um aumento do índice mitótico, coloração da proteína argirofílica nuclear e células tumorais ciclando em tumores em crescimento, respectivamente. Todos os três marcadores de proliferação celular foram capazes de distinguir o TVTC em ambas as fases de evolução. O anticorpo monoclonal MIB 1 foi o melhor na avaliação morfológica, de crescimento e regressão do TVTC. Isto resultou em um valor maior que a contagem de AgNOR e do índice mitótico. A imunomarcação com MIB1 foi o parâmetro mais efetivo da atividade proliferativa. No entanto, só foi observada uma correlação positiva entre a contagem de mitose e a AgNOR.(AU)

Cell proliferation markers in the transplanted canine transmissible venereal tumor / Marcadores de proliferação celular no tumor venéreo transmissível canino transplantado

Adult male mongrel dogs were subcutaneously transplanted with the canine transmissible venereal tumor (TVT) on the hypogastric region. Twelve specimens of tumors were collected, half during the proliferative phase and the other half during the regressive phase. Fragments of the tumor were fixed in 10 percent buffered formalin and routinely processed for light microscopy. Sections of 4µm were stained by Schorr or AgNOR or either immunostained for MIB1 (Ki67). Schorr stain, AgNOR and MIB1 showed an increased proliferative activity through mitotic index, nuclear argyrophilic protein stain and cycling tumoral cells in the growing tumors, respectively. All of the three cell proliferation markers were able to distinguish the TVT in both evolution phases. MIB1 monoclonal antibody was the best in the morphologic evaluation of growth and regression of TVT. This resulted in higher values than AgNORs counting and mitotic index. MIB1 immunostaining was the most effective parameter of the proliferative activity of TVT. However, a significant correlation has been detected only between mitosis counting and AgNORs.

Cães machos, adultos, mestiços, foram transplantados com células do tumor venéreo transmissível canino (TVTC), na região hipogástrica. Foram coletados doze espécimes do TVTC, sendo metade durante a fase proliferativa e metade durante a fase regressiva. Fragmentos do tumor foram fixados em formol a 10 por cento, tamponado e processado rotineiramente para microscopia de luz. Secções de 4µm foram coradas pelo Shorr, ou pela AgNOR, ou ainda, imunocorado para MIB 1 (Ki67). As colorações pelo Shorr, AgNOR, ou MIB 1 mostraram um aumento do índice mitótico, coloração da proteína argirofílica nuclear e células tumorais ciclando em tumores em crescimento, respectivamente. Todos os três marcadores de proliferação celular foram capazes de distinguir o TVTC em ambas as fases de evolução. O anticorpo monoclonal MIB 1 foi o melhor na avaliação morfológica, de crescimento e regressão do TVTC. Isto resultou em um valor maior que a contagem de AgNOR e do índice mitótico. A imunomarcação com MIB1 foi o parâmetro mais efetivo da atividade proliferativa. No entanto, só foi observada uma correlação positiva entre a contagem de mitose e a AgNOR.

Cell proliferation markers in the transplanted canine transmissible venereal tumor

Adult male mongrel dogs were subcutaneously transplanted with the canine transmissible venereal tumor (TVT) on the hypogastric region. Twelve specimens of tumors were collected, half during the proliferative phase and the other half during the regressive phase. Fragments of the tumor were fixed in 10% buffered formalin and routinely processed for light microscopy. Sections of 4µm were stained by Schorr or AgNOR or either immunostained for MIB1 (Ki67). Schorr stain, AgNOR and MIB1 showed an increased proliferative activity through mitotic index, nuclear argyrophilic protein stain and cycling tumoral cells in the growing tumors, respectively. All of the three cell proliferation markers were able to distinguish the TVT in both evolution phases. MIB1 monoclonal antibody was the best in the morphologic evaluation of growth and regression of TVT. This resulted in higher values than AgNORs counting and mitotic index. MIB1 immunostaining was the most effective parameter of the proliferative activity of TVT. However, a significant correlation has been detected only between mitosis counting and AgNORs.

Cães machos, adultos, mestiços, foram transplantados com células do tumor venéreo transmissível canino (TVTC), na região hipogástrica. Foram coletados doze espécimes do TVTC, sendo metade durante a fase proliferativa e metade durante a fase regressiva. Fragmentos do tumor foram fixados em formol a 10%, tamponado e processado rotineiramente para microscopia de luz. Secções de 4µm foram coradas pelo Shorr, ou pela AgNOR, ou ainda, imunocorado para MIB 1 (Ki67). As colorações pelo Shorr, AgNOR, ou MIB 1 mostraram um aumento do índice mitótico, coloração da proteína argirofílica nuclear e células tumorais ciclando em tumores em crescimento, respectivamente. Todos os três marcadores de proliferação celular foram capazes de distinguir o TVTC em ambas as fases de evolução. O anticorpo monoclonal MIB 1 foi o melhor na avaliação morfológica, de crescimento e regressão do TVTC. Isto resultou em um valor maior que a contagem de AgNOR e do índice mitótico. A imunomarcação com MIB1 foi o parâmetro mais efetivo da atividade proliferativa. No entanto, só foi observada uma correlação positiva entre a contagem de mitose e a AgNOR.

Phosphatidylinositol 3-kinase-δ up-regulates L-type Ca2+ currents and increases vascular contractility in a mouse model of type 1 diabetes.

BACKGROUND AND PURPOSE: Vasculopathies represent the main cause of morbidity and mortality in diabetes. Vascular malfunctioning in diabetes is associated with abnormal vasoconstriction and Ca(2+) handling by smooth muscle cells (SMC). Phosphatidylinositol 3-kinases (PI3K) are key mediators of insulin action and have been shown to modulate the function of voltage-dependent L-type Ca(2+) channels (Ca(V) 1.2). In the present work, we investigated the involvement of PI3K signalling in regulating Ca(2+) current through Ca(V) 1.2 (I(Ca,L) ) and vascular dysfunction in a mouse model of type I diabetes. EXPERIMENTAL APPROACH: Changes in isometric tension were recorded on myograph. Ca(2+) currents in freshly dissociated mice aortic SMCs were measured using the whole-cell patch-clamp technique. Antisense techniques were used to knock-down the PI3Kδ isoform. KEY RESULTS Contractile responses to phenylephrine and KCl were strongly enhanced in diabetic aorta independent of a functional endothelium. The magnitude of phenylephrine-induced I(Ca,L) was also greatly augmented. PI3Kδ expression, but not PI3Kα, PI3Kß, PI3Kγ, was increased in diabetic aortas and treatment of vessels with a selective PI3Kδ inhibitor normalized I(Ca,L) and contractile response of diabetic vessels. Moreover, knock-down of PI3Kδin vivo decreased PI3Kδ expression and normalized I(Ca,L) and contractile response of diabetic vessels ex vivo. CONCLUSIONS AND IMPLICATIONS: Phosphatidylinositol 3-kinase δ was essential to the increased vascular contractile response in our model of type I diabetes. PI3Kδ signalling was up-regulated and most likely accounted for the increased I(Ca,L,) leading to increased vascular contractility. Blockade of PI3Kδ may represent a novel therapeutic approach to treat vascular dysfunction in diabetic patients.

Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats.

AIMS: Thiamine is an important cofactor present in many biochemical reactions, and its deprivation can lead to heart dysfunction. Little is known about the influence of thiamine deprivation on the electrophysiological behavior of the isolated heart cells and information about thiamine deficiency in heart morphology is controversial. Thus, we decided to investigate the major repolarizing conductances and their influence in the action potential (AP) waveform as well as the changes in the heart structure in a set of thiamine deficiency in rats. MAIN METHODS: Using the patch-clamp technique, we investigated inward (I(K1)) and outward K(+) currents (I(to)), T-type and L-type Ca(2+) currents and APs. To evaluate heart morphology we used hematoxylin and eosin in transversal heart sections. KEY FINDINGS: Thiamine deficiency caused a marked decrease in left ventricle thickness, cardiomyocyte number, cell length and width, and membrane capacitance. When evaluating I(to) we did not find difference in current amplitude; however an acceleration of I(to) inactivation was observed. I(K1) showed a reduction in the amplitude and slope conductance, which implicated a less negative resting membrane potential in cardiac myocytes isolated from thiamine-deficient rats. We did not find any difference in L-type Ca(2+) current density. T-type Ca(2+) current was not observed. In addition, we did not observe significant changes in AP repolarization. SIGNIFICANCE: Based on our study we can conclude that thiamine deficiency causes heart hypotrophy and not heart hypertrophy. Moreover, we provided evidence that there is no major electrical remodeling during thiamine deficiency, a feature of heart failure models.

Cellular proliferation, differentiation and apoptosis in polyether-polyurethane sponge implant model in mice.

The integration of implanted material to host organism requires spatial and temporal organization of several cellular processes, such as proliferation, differentiation and apoptosis. Despite the clinical relevance of these processes, there is little information regarding the sequence of such events in synthetic matrices. Here, we present a combination of techniques used to characterize the fibrovascular response in subcutaneous polyether-polyurethane sponge implants in mice at days 4, 7, 10 and 14 postimplantation. The AgNOR technique was modified and used as a surrogate marker for proliferating and activated cells invading the implant. The number of AgNOR-stained cells increased progressively from day 4 (606+/-76) to day 14 (2146+/-71) postimplantation. The number of TUNEL-positive (apoptotic index) cells also increased progressively from day 4 (459+/-40) to day 14 (1157+/-119) postimplantation. However, the ratio of TUNEL-labeled/proliferating cells had its highest peak in the early phase of the process remaining stable until day 14. Using Picrosirius staining it was shown that thin collagen increased from day 4, peaking at day 10 and falling markedly at day 14, whereas dense collagen increased progressively during the whole period. These experiments hold potential to investigate not only distinct phases of tissue repair induced by synthetic matrices but also to study underlying mechanisms involved.

Morphometric analysis of the thymus of puppies infected with the Snyder Hill Strain of canine distemper virus / Análise morfométrica do timo de cães filhotes infectados com a amostra Snyder Hill do vírus de cinomose canina

The thymic morphometry analysis was used for determining apoptosis and atrophy of the thymus of eight puppies inoculated with canine distemper virus (CDV). Three healthy dogs were used as negative controls. Sections, 5µm thick, were stained by HE and Shorr, and the latter were evaluated by morphometry. CDV nucleoprotein was detected by immunohistochemistry. Morphometric results confirmed lymphoid hypotrophy in CDV inoculated dog thymuses, more stroma, less parenchyma and higher apoptotic index/field than negative control (not inoculated) puppies. Apoptosis plays a role in the mechanism of thymus atrophy that develops in canine distemper.(AU)

Determinaram-se a apoptose e a atrofia no timo de oito cães novos, inoculados experimentalmente com o vírus de cinomose. Três cães saudáveis foram usados como controle negativo. Secções coradas pelo Shorr foram avaliadas por morfometria. A nucleoproteína viral foi detectada por imunoistoquímica. Os resultados morfométricos confirmaram a hipotrofia e mostraram que o timo dos cães inoculados tinha mais estroma, menos parênquima e maior índice apoptótico/campo que o dos animais-controle. Pode–se concluir que a apoptose desempenha importante papel no mecanismo de hipotrofia tímica que se desenvolve na cinomose.(AU)

Morphometric analysis of the thymus of puppies infected with the Snyder Hill Strain of canine distemper virus / Análise morfométrica do timo de cães filhotes infectados com a amostra Snyder Hill do vírus de cinomose canina

The thymic morphometry analysis was used for determining apoptosis and atrophy of the thymus of eight puppies inoculated with canine distemper virus (CDV). Three healthy dogs were used as negative controls. Sections, 5æm thick, were stained by HE and Shorr, and the latter were evaluated by morphometry. CDV nucleoprotein was detected by immunohistochemistry. Morphometric results confirmed lymphoid hypotrophy in CDV inoculated dog thymuses, more stroma, less parenchyma and higher apoptotic index/field than negative control (not inoculated) puppies. Apoptosis plays a role in the mechanism of thymus atrophy that develops in canine distemper.

Determinaram-se a apoptose e a atrofia no timo de oito cães novos, inoculados experimentalmente com o vírus de cinomose. Três cães saudáveis foram usados como controle negativo. Secções coradas pelo Shorr foram avaliadas por morfometria. A nucleoproteína viral foi detectada por imunoistoquímica. Os resultados morfométricos confirmaram a hipotrofia e mostraram que o timo dos cães inoculados tinha mais estroma, menos parênquima e maior índice apoptótico/campo que o dos animais-controle. Pode-se concluir que a apoptose desempenha importante papel no mecanismo de hipotrofia tímica que se desenvolve na cinomose.

Apoptosis in the placentomo of pregnant goats experimentally intoxicated with cipó-preto - Tetrapterys multiglandulosa / Apoptose no placentomo de cabras gestantes intoxicadas experimentalmente com cipó-preto - Tetrapterys multiglandulosa

Estudou-se a ocorrência de apoptose nos placentomos de cabras gestantes intoxicadas experimentalmente com o cipó-preto (Tetrapterys multiglandulosa A.Juss.). Analisou-se morfometricamente a intensidade do processo de apoptose nas células trofoblásticas em cabras controle (grupo III) e em animais submetidos a diferentes dosagens (grupos I e II) de cipó-preto. O grupo I foi composto por quatro cabras gestantes que receberam 10gramas/kg de peso vivo de folhas verdes da referida planta. No grupo II, quatro cabras gestantes receberam 20 gramas/kg de peso vivo de folhas verdes. A quantificação morfométrica da apoptose demonstrou que nas cabras tratadas a apoptose ocorreu com maior intensidade quando comparada com a obtida em animais do grupo-controle. Diferentes dosagens da planta (10 e 20g/kg PV) não foram um fator determinante para a maior ou menor ocorrência de apoptose, apesar de acarretar morte fetal e subseqüente aborto em momentos diferentes. A intensa apoptose em fase ainda inicial da gestação compromete funções normais da placenta, possibilitando uma explicação para a morte fetal e aborto observadas. Conclui-se que a T. multiglandulosa é tóxica para cabras gestantes nas doses de 10 e 20g/kg peso vivo ingeridas durante seis e duas semanas, respectivamente.(AU)

The role of apoptosis in the pathogenesis of placental dysfunction was studied in pregnant goats experimentally intoxicated with cipó-preto (Tetrapterys multiglandulosa A.Juss.). Twelve animals were distributed in three groups, each one with four pregnant goats. Group I was fed 10g/kg of live weight and group II was fed 20g/kg of live weight and group III was used as control. Although apoptosis occurred in placental cells of all the animals, morphometric analyses demonstrated that goats fed with cipó-preto showed higher apoptotic indices than controls. However no difference could be detected between groups I and II. Different amounts of the plant were not a determinant factor to increase the intensity of apoptosis in the placental tissue, although the final result was fetal death and/or abortion at different moments. Intense apoptosis during the early stage of pregnancy may be detrimental for the normal development and function of the placenta and may help to explain the fetal death and abortion observed. Therefore Tetrapterys multiglandulosa is toxic to pregnant goats when ingested at 10 or 20g/kg of live weight during six or two weeks, respectively.(AU)

Apoptose no placentomo de cabras gestantes intoxicadas experimentalmente com cipó-preto - Tetrapterys multiglandulosa / Apoptosis in the placentomo of pregnant goats experimentally intoxicated with cipó-preto - Tetrapterys multiglandulosa

Estudou-se a ocorrência de apoptose nos placentomos de cabras gestantes intoxicadas experimentalmente com o cipó-preto (Tetrapterys multiglandulosa A.Juss.). Analisou-se morfometricamente a intensidade do processo de apoptose nas células trofoblásticas em cabras controle (grupo III) e em animais submetidos a diferentes dosagens (grupos I e II) de cipó-preto. O grupo I foi composto por quatro cabras gestantes que receberam 10gramas/kg de peso vivo de folhas verdes da referida planta. No grupo II, quatro cabras gestantes receberam 20 gramas/kg de peso vivo de folhas verdes. A quantificação morfométrica da apoptose demonstrou que nas cabras tratadas a apoptose ocorreu com maior intensidade quando comparada com a obtida em animais do grupo-controle. Diferentes dosagens da planta (10 e 20g/kg PV) não foram um fator determinante para a maior ou menor ocorrência de apoptose, apesar de acarretar morte fetal e subseqüente aborto em momentos diferentes. A intensa apoptose em fase ainda inicial da gestação compromete funções normais da placenta, possibilitando uma explicação para a morte fetal e aborto observadas. Conclui-se que a T. multiglandulosa é tóxica para cabras gestantes nas doses de 10 e 20g/kg peso vivo ingeridas durante seis e duas semanas, respectivamente.