Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18.

Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-ß and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.

Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

FSH up-regulates angiogenic factors in luteal cells of buffaloes.

Follicle-stimulating hormone has been widely used to induce superovulation in buffaloes and cows and usually triggers functional and morphologic alterations in the corpus luteum (CL). Several studies have shown that FSH is involved in regulating vascular development and that adequate angiogenesis is essential for normal luteal development. Angiogenesis is regulated by many growth factors, of which vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) have an established central role. Therefore, we have used a combination of in vitro and in vivo studies to assess the effects of FSH on the expression of VEGF and FGF2 and their receptors in buffalo luteal cells. The in vivo model consisted of 12 buffalo cows, divided into control (n = 6) and superovulated (n = 6) groups, and CL samples were collected on day 6 after ovulation. In this model, we analyzed the gene and protein expression of FGF2 and its receptors and the protein expression of VEGFA systems with the use of real-time PCR, Western blot analysis, and immunohistochemistry. In the in vitro model, granulosa cells were collected from small follicles (diameter, 4-6 mm) of buffaloes and cultured for 4 d in serum-free medium with or without FSH (10 ng/mL). To induce in vitro luteinization, LH (250 ng/mL) and fetal bovine serum (10%) were added to the medium, and granulosa cells were maintained in culture for 4 d more. The progesterone concentration in the medium was measured at days 4, 5, and 8 after the beginning of cell culture. Cells were collected at day 8 and subjected to real-time PCR, Western blot analysis, and immunofluorescence for assessment of the expression of FGF2, VEGF, and their receptors. To address the percentage of steroidogenic and growth factor-expressing cells in the culture, flow cytometry was performed. We observed that in superovulated buffalo CL, the FGF2 system mRNA expression was decreased even as protein expression was increased and that the VEGF protein was increased (P < 0.05). In vitro experiments with granulosa cells showed an increase in the mRNA expression of VEGF and FGF2 and its receptors 1 and 2 and protein expression of VEGF, kinase insert domain receptor, FGF receptor 2, and FGF receptor 3 in cells treated with FSH (P < 0.05), in contrast to the in vivo experiments. Moreover, the progesterone production by FSH-treated cells was elevated compared with untreated cells (P < 0.05). Our findings indicate that VEGF, FGF2, and their receptors were differentially regulated by FSH in vitro and in vivo in buffalo luteal cells, which points toward a role of CL environment in modulating cellular answers to gonadotropins.

Bronchial hyperresponsiveness and analysis of induced sputum cells in Crohn's disease.

With the aim of investigating the presence of latent inflammatory process in the lungs of patients with Crohn's disease, 15 patients with Crohn's disease were evaluated by spirometry, the methacholine challenge test, induced sputum, and skin tests for inhaled antigens. Serum IgE, erythrocyte sedimentation rate and hematocrit were also determined. The patients were compared with 20 healthy controls by the Mann-Whitney and Fisher exact tests. Their respiratory physical examination was normal. None had a personal or family history of clinical atopy. None had a previous history of pulmonary disease, smoking or toxic bronchopulmonary exposure. None had sinusitis, migraine, diabetes mellitus, or cardiac failure. Four (26.6%) of the patients with Crohn's disease had a positive methacholine challenge test whereas none of the 20 controls had a positive methacholine test (P = 0.026, Fisher exact test). Patients with Crohn's disease had a higher level of lymphocytes in induced sputum than controls (mean 14.59%, range 3.2-50 vs 5.46%, 0-26.92%, respectively; P = 0.011, Mann-Whitney test). Patients with Crohn's disease and a positive methacholine challenge test had an even higher percentage of lymphocytes in induced sputum compared with patients with Crohn's disease and a negative methacholine test (mean 24.88%, range 12.87-50 vs 10.48%, 3.2-21.69%; P = 0.047, Mann-Whitney test). The simultaneous findings of bronchopulmonary lymphocytosis and bronchial hyperresponsiveness in patients with Crohn's disease were not reported up to now. These results suggest that patients with Crohn's disease present a subclinical inflammatory process despite the absence of pulmonary symptoms.

Bronchial hyperresponsiveness and analysis of induced sputum cells in Crohn's disease

With the aim of investigating the presence of latent inflammatory process in the lungs of patients with Crohn's disease, 15 patients with Crohn's disease were evaluated by spirometry, the methacholine challenge test, induced sputum, and skin tests for inhaled antigens. Serum IgE, erythrocyte sedimentation rate and hematocrit were also determined. The patients were compared with 20 healthy controls by the Mann-Whitney and Fisher exact tests. Their respiratory physical examination was normal. None had a personal or family history of clinical atopy. None had a previous history of pulmonary disease, smoking or toxic bronchopulmonary exposure. None had sinusitis, migraine, diabetes mellitus, or cardiac failure. Four (26.6 percent) of the patients with Crohn's disease had a positive methacholine challenge test whereas none of the 20 controls had a positive methacholine test (P = 0.026, Fisher exact test). Patients with Crohn's disease had a higher level of lymphocytes in induced sputum than controls (mean 14.59 percent, range 3.2-50 vs 5.46 percent, 0-26.92 percent, respectively; P = 0.011, Mann-Whitney test). Patients with Crohn's disease and a positive methacholine challenge test had an even higher percentage of lymphocytes in induced sputum compared with patients with Crohn's disease and a negative methacholine test (mean 24.88 percent, range 12.87-50 vs 10.48 percent, 3.2-21.69 percent; P = 0.047, Mann-Whitney test). The simultaneous findings of bronchopulmonary lymphocytosis and bronchial hyperresponsiveness in patients with Crohn's disease were not reported up to now. These results suggest that patients with Crohn's disease present a subclinical inflammatory process despite the absence of pulmonary symptoms.

Cyanogenic potential of cassava flour: field trial in Mozambique of a simple kit.

The cyanogenic potential (ppm HCN equivalents) of 80 samples of cassava flour (obtained from the Mujocojo and Terrene-A areas of Nampula Province and the markets of Nampula City in Mozambique) were determined using a new simple kit, based on the use of picric acid paper (Egan et al., 1997). The kit is compact, requires only a small amount of water and is very simple to use in the field. Comparison with the results of a semi-quantitative method shows a mean deviation between the two methods of 20% (SD 12%). All samples fitted a single population distribution with a mean value of 45 ppm HCN equivalents (SD 37). Two maxima were observed in the distribution curve at 11-20 and 41-50 ppm. Five samples exceeded 100 ppm with two values of 200 ppm. The WHO safe level for cyanogens in cassava flour is 10 ppm. The lowest levels (2 and 6 ppm) were obtained from cassava flour prepared from sweet cassava. Over 76 samples the mean value of the cyanogenic potential of cassava flour produced by heap fermentation is only one half as large as that produced by sun-drying (P < 0.005). Interventions needed to reduce cyanogen levels are (1) improvements in processing methods, such as replacement of sun-drying by heap fermentation, (2) introduction of additional vegetables, pulses and fruit to alleviate the monotonous cassava diet of the people and (3) introduction of high-yielding, disease-resistant, low-cyanide cultivars.

Sintomas respiratórios e testes espirográficos dos funcionários de uma pedreira do Rio de Janeiro / Respiratory symptoms and spirometric tests of the quarry workers Rio de Janeiro

Foram investigados os sintomas e antecedentes respiratórios, bem como os resultados das provas espirográficas, de 72 dos 86 (83,7 por cento) funcionários de uma pedreira de porte médio do Rio de Janeiro. Setenta e um (98,6 por cento) eram homens, e a média das idades foi de 36,2 + ou - 9,3 anos (20 a 65 anos). Quarenta e dois (58,3 por cento) tinham ocupaçöes consideradas muito expostas ao pó de pedra e 30 (44,7 por cento) pouco expostas. Quarenta (55,6 por cento) eram fumantes, 20 (27,8 por cento) näo fumantes e 12 (16,7 por cento) ex-fumantes. Foi utilizado um questionário padronizado baseado em modelos internacionais e adaptados a condiçöes brasileiras. Houve elevada freqüência de sintomas, com exceçäo da dispnéia: 47 (65,3 por cento) referiram um ou mais sintomas, dos quais 31,9 por cento referiram tosse, 41,7 por cento expectoraçäo, 9,7 por cento dispnéia e 33,3 por cento chiado torácico. Os sintomas se associaram principalmente à presença de antecedentes de doenças respiratórias, havendo também associaçäo com o tabagismo. Um ou outro desses fatores explicaram os sintomas. Näo houve associaçäo com a maior exposiçäo ao pó de pedra, qua näo explicou os sintomas. As provas espirográficas foram realizadas com o espirógrafo de campânula de 6 litros, observando-se discreta reduçäo das médias da capacidade vital forçada (CVF) (89,9 + ou - 11,2 por cento do teórico), do volume de expiraçäo forçada do 1§ segundo (VEF1) (90,1 + ou - 12,9 por cento) e do fluxo expiratório forçado entre 25 por cento e 75 por cento da CVF (FEF 25-75) (92,9 + ou - 32,7 por cento) em relaçäo às médias esperadas numa amostra populacional näo selecionada. Contudo, as proporçöes de valores subnormais foram comparáveis às de amostras näo selecionadas da literatura nacional e internacional. Näo houve influência estatisticamente significativa da maior exposiçäo ao pó de pedra, antecedentes respiratórios e tabagismo sobre as médias dos resultados espirográficos. Concluímos que, neste grupo de funcionários de uma pedreira, näo se demonstrou associaçäo entre a maior exposiçäo ao pó de pedra e os sintomas respiratórios, ou influência dessa exposiçäo sobre as médias dos resultados espirográficos. Com relaçäo aos antecedentes respiratórios e tabagismo, houve associaçäo com os sintomas mas näo influência sobre as médias dos resultados espirográficos.

[Respiratory symptoms and spirometric tests of quarry workers in Rio de Janeiro]. / Sintomas respiratórios e testes espirográficos dos funcionários de uma pedreira do Rio de Janeiro.

Respiratory symptoms and past history of chest disease as well as spirometric tests were investigated in 72 of 86 (83.7%) employees of a middle sized quarry in Rio de Janeiro, Brazil. Seventy one were men (98.6%), and the mean age was 36.2 +/- 9.3 years (20-65). Forty two (58.3%) had occupations considered as highly exposed to dust, and 30 (44.7%) were considered as lightly exposed. Forty (55.6%) were smokers, 20 (27.8%) never had been smokers and 12 (16.7%) were former smokers. Symptoms and past history were investigated with a questionnaire based on international models, and adapted for the Brazilian public. A high prevalence of symptoms (except for dyspnea) was noted: 47 (65.3%) had one or more symptoms-there was cough in 31.9%, expectoration in 41.7% dyspnea in 9.7% and wheezing in 33.3%. The symptoms were found to be associated mostly with a past history of chest disease, and also with smoking, factors which explained, in part, the presence of the symptoms. No association with a higher exposure to dust was found. The exposure to dust did not explain the symptoms. The spirometric tests were performed on a 6 liter bell spirometer. The means of the parameters were lower than one would expect in a non selected population sample--89.9 +/- 11.2% of predicted for the Forced Vital Capacity (FVC), 90.1 +/- 12.9% for the Forced Expiratory Volume in 1 second (FEV1) and 92.9 +/- 32.7% for the Forced Expiratory Flow between 25% and 75% of the FVC (FEF25-75). However, the proportions of abnormal results were comparable to the reported ones from unselected samples. There was no statistically significant influence of higher dust exposure, past history or smoking on the means of the results. It is concluded that, in this sample of quarry employees, no association between respiratory symptoms or spirometric results and dust exposure could be demonstrated. There was an association between the symptoms, but not with the spirometric results, and a past history of chest disease, as well as with smoking.

Quadro espirografico na fase assintomatica da asma bronquica. / Spirometric study in asymptomatic period of bronchial asthma

O estudo espirografico de 67 asmaticos totalmente assintomaticos e com o exame fisico normal, atendidos consecutivamente e sem carater de selecao, a nao ser exclusao de doencas concomitantes, mostrou que a obstrucao persistia em 76,1%. Dos l3 com espirograma normal, 9 revelaram resposta significativa apos broncodilatacao. Reforca-se, assim, a impressao de que ha na asma um exame do tonus broncomotor independente de fatores desencadeantes. Em 49,2% a obstrucao esteve presente ao longo de toda a expiracao e em apenas 26,8% so na parte final. A reducao da capacidade vital ocorreu em apenas 11,9%, sempre aliada a obstrucao. A prova broncodialtadora revelou melhora da obstrucao em 68,6% dos casos, e, no grupo todo, o aumento foi significativo em todos os parametros estudados (p < 0,05). No nivel de significancia de 5%, nao houve diferenca entre asmaticos nao-fumantes e asmaticos fumantes ou exfumantes, quer nos resultados pre-broncodilatacao, quer na resposta broncodilatadora, a excecao unica da capacidade vital forcada, em que houve aumento significativamente maior entre fumantes e ex-fumantes (p < 0,05)