The Tumor Microenvironment: An Introduction to the Development of Microfluidic Devices.

The tumor microenvironment (TME) is like the Referee of a soccer match who has constant eyes on the activity of all players, such as cells, acellular stroma components, and signaling molecules for the successful completion of the game, that is, tumorigenesis. The cooperation among all the "team members" determines the characteristics of tumor, such as the hypoxic and acidic niche, stiffer mechanical properties, or dilated vasculature. Like in soccer, each TME is different. This heterogeneity makes it challenging to fully understand the intratumor dynamics, particularly among different tumor subpopulations and their role in therapeutic response or resistance. Further, during metastasis, tumor cells can disseminate to a secondary organ, a critical event responsible for approximately 90% of the deaths in cancer patients. The recapitulation of the rapidly changing TME in the laboratory is crucial to improve patients' prognosis for unraveling key mechanisms of tumorigenesis and developing better drugs. Hence, in this chapter, we provide an overview of the characteristic features of the TME and how to model them, followed by a brief description of the limitations of existing in vitro platforms. Finally, various attempts at simulating the TME using microfluidic platforms are highlighted. The chapter ends with the concerns that need to be addressed for designing more realistic and predictive tumor-on-a-chip platforms.

Quantitative Viral Outgrowth Assay to Measure the Functional SIV Reservoir in Myeloid Cells.

The role of CD4+ T cells in HIV infection and the latent reservoir, that is, latently infected cells that harbor replication competent virus, has been rigorously assessed. We have previously reported a quantitative viral outgrowth assay (QVOA) for SIV that demonstrated the frequency of latently infected CD4+ T cells is approximately 1 in a million cells, similar to that of HIV infected individuals on ART. However, the frequency of productively infected monocytes in blood and macrophages in tissues has not been similarly studied. Myeloid cells are infected during acute HIV and SIV infection; however, unlike lymphocytes, they are resistant to the cytopathic effects of the virus. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a stable viral reservoir. A better understanding of the number of productively infected macrophages is critical to understanding the role of infected myeloid cells as a viral reservoir. In order to assess the functional latent reservoir. we have developed specific QVOAs for monocytes in blood, and macrophages in spleen, BAL and brain, which are described in detail in this chapter.

Modulation of inflammation by anti-TNF α mAb-dendrimer nanoparticles loaded in tyramine-modified gellan gum hydrogels in a cartilage-on-a-chip model.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease characterized by joint inflammation. Since the inflammatory condition plays an important role in the disease process, it is important to develop and test new therapeutic approaches that specifically target and treat joint inflammation. In this study, a human 3D inflammatory cartilage-on-a-chip model was established to test the therapeutic efficacy of anti-TNFα mAb-CS/PAMAM dendrimer NPs loaded-Tyramine-Gellan Gum in the treatment of inflammation. The results showed that the proposed therapeutic approach applied to the human monocyte cell line (THP-1) and human chondrogenic primary cells (hCH) cell-based inflammation system revealed an anti-inflammatory capacity that increased over 14 days. It was also possible to observe that Coll type II was highly expressed by inflamed hCH upon the culture with anti-TNF α mAb-CS/PAMAM dendrimer NPs, indicating that the hCH cells were able maintain their biological function. The developed preclinical model allowed us to provide more robust data on the potential therapeutic effect of anti-TNF α mAb-CS/PAMAM dendrimer NPs loaded-Ty-GG hydrogel in a physiologically relevant model.

Direct monitoring of breast and endometrial cancer cell epigenetic response to DNA methyltransferase and histone deacetylase inhibitors.

DNA methylation and histone deacetylation are key epigenetic processes involved in normal cellular function and tumorigenesis. Therapeutic strategies based on DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors are currently in use and under development for the treatment of cancers. Genome-wide DNA methylation profiling has been proposed for use in disease diagnosis, and histone modification profiling for disease stratification will follow suit. However, whether epigenome sequencing technologies will be feasible for rapid clinic diagnosis and patient treatment monitoring remains to be seen, and alternative detection technologies will almost certainly be needed. Here we used electrochemical impedance spectroscopy (EIS) employing a graphene-based screen-printed electrode system to directly measure global DNA methylation and histone H3 acetylation to compare non-cancer and breast cancer cell lines. We demonstrated that whilst global methylation was not useful as a differential marker in the cellular systems tested, histone H3 acetylation was effective at higher chromatin levels. Using breast and endometrial cancer cell models, EIS was then used to monitor cellular responses to the DNMT and HDAC inhibitors 5-Aza-2'-deoxycytidine and suberoylanilide hydroxamic acid in vitro, and proved very effective at detecting global cellular responses to either treatment, indicating that this approach could be useful in following treatment response to epigenetic drugs. Moreover, this work reports the first combined analysis of two epigenetic markers using a unified graphene-based biosensor platform, demonstrating the potential for multiplex analysis of both methylation and acetylation on the same sample.

Chemically sulfated galactomannan from Dimorphandra gardneriana seed: characterization and toxicity evaluation.

Dimorphandra gardneriana galactomannan (DG) was sulfated in pyridine:formamide using chlorosulfonic acid as the sulfation agent. The degree of substitution was 0.32, determined from the sulfur percentage. Confirmation of sulfation was obtained by FTIR spectroscopy through the presence of an asymmetrical SO stretching vibration at 1,259 cm(-1). NMR data showed that the sulfation occurred on primary hydroxyl groups. NMR and GPC data indicate degradation during reaction with elimination of galactose. At the maximum tested concentration of 1,000 µg/mL, unmodified DG polysaccharide did not show a statistically significant cytotoxicity in Vero cells by the MTT method. Therefore, the CC50>1,000 µg/mL obtained for the sulfated polysaccharides from D. gardneriana in Vero cells point to its lower cytotoxicity than the sulfated galactomannan from Mimosa scabrella.

Avaliação preliminar in vivo do efeito hipocolesterolêmico do polvilho da fruta-de-lobo (Solanum lycocarpum A. St.-Hil. ) / Preliminary evaluation of in vivo hypocholesterolemic effect of the flour of fruit-of-wolf (Solanum lycocarpum A. St.-Hil. )

O polvilho da fruta-de-lobo é um produto extraído da polpa da fruta-de-lobo verde (Solanum lycocarpum A. St.-Hil.), popularmente utilizado. Pouco se conhece a respeito desse polvilho, mas são atribuídos a ele vários efeitos terapêuticos, dentre eles a redução do colesterol. Este trabalho teve o objetivo de verificar a ação da administração do polvilho da fruta-de-lobo sobre animais hipercolesterolêmico. Utilizou-se 24 ratos Wistar fêmeas distribuídas em delineamento ao acaso, em três grupos contendo 8 animais em cada grupo. Estes grupos foram definidos como: controle normal (CN), controle hipocolesterolêmico (CH) e hipocolesterolêmico tratado (HT). O grupo CN recebeu dieta comercial, os grupo CH e HT receberam a dieta comercial enriquecida de colesterol e ácido cólico e o grupo HT recebeu também, 100 mg de polvilho da fruta-lobo, diariamente, por sonda orogástrica. O experimento teve uma duração de 6 semanas onde se avaliou o colesterol total sérico semanalmente, peso dos animais semanalmente e o consumo diário de ração. Ao término do experimento, foram avaliados os seguintes parâmetros: frações séricas de colesterol HDL, LDL+VLDL, peso do fígado, colesterol total hepático, lipídeos totais hepático e lâminas de microscopia foram feitas para avaliação dos hepatócitos. Não houve diferença significativa em relação ao peso corporal dos animais, ao consumo da dieta e nas análises de colesterol total sérico entre os grupos estudados. Avaliando-se a relação VLDL +LDL/HDL, os níveis encontrados no grupo HT foram significativamente menores que o grupo CH. Já em relação ao colesterol hepático, o grupo HT mostrou níveis menores de colesterol que o CH. Observou-se nos lipídeos hepáticos que não houve diferença significativa entre os grupo CH e HT, e no peso do fígado houve diferença significativa entre os grupos avaliados. Em relação à microscopia, os grupos hipercolesterolêmicos apresentaram discreta vacuolização no citoplasma dos hepatócitos. Concluiu-se, que o polvilho da fruta-de-lobo não influenciou o colesterol sérico dos animais, entretanto, reduziu os níveis de colesterol hepático.

The fruit-of-wolf flour is a product extracted from the pulp of green fruit-of-wolf (Solanum lycocarpum A. St .- Hil.) and is commonly used. Little is known about this flour, but several therapeutic effects, including cholesterol reduction, are attributed to it. This study aimed to verify the action of the administration of fruit-of-wolf flour to hypercholesterolemic animals. We used 24 female Wistar rats allocated in randomized design to three groups containing 8 animals each. These groups were defined as normal control (CN), hypocholesterolemic control (CH) and hypocholesterolemic treated rats (HT). The CN group received a commercial diet, while the CH and the HT group received the commercial diet enriched with cholesterol and cholic acid; the HT group also received 100 mg of fruit-of-wolf flour, daily, by orogastric tube. The experiment lasted for six weeks and the following characteristics were evaluated: weekly total serum cholesterol, weekly weight of animals and daily food intake. At the end of the experiment, we assessed the following parameters: serum cholesterol fractions HDL, LDL + VLDL, liver weight, liver total cholesterol, liver total lipids and microscopic slides were prepared for the evaluation of hepatocytes. There was no significant difference in body weight of animals, diet consumption and analysis of serum total cholesterol among the studied groups. Assessing the relationship VLDL + LDL / HDL, the levels found for the HT group were significantly lower than those for the CH group. As regards liver cholesterol, the HT group showed lower cholesterol levels than the CH group. For liver lipids there was no significant difference between the CH and the HT group, and for liver weight there was no significant difference among the studied groups. As to microscopy, the hypercholesterolemic groups showed slight vacuolization in the cytoplasm of hepatocytes. It was concluded that fruit-of-wolf flour did not influence the serum cholesterol of animals but reduced the levels of liver cholesterol.

Atividade das glicosidases na presença de chá verde e de chá preto / Activity of glycosidases in the presence of green tea and black tea

Várias plantas têm sido consideradas produtos terapêuticos, dentre elas destacam-se os chás verde e preto, popularmente utilizados para controle da hiperglicemia e obesidade. Objetivou-se neste trabalho avaliar o potencial inibitório sobre as enzimas α-amilase, α e β-glicosidases e o teor de compostos fenólicos do chá verde e do chá preto. O teor de compostos fenólicos encontrados foram de 80,8 ± 0,43 mg g-1 no chá preto e 32,0 ± 0,12 mg g-1 no chá verde. O chá verde e o chá preto, em condições de consumo, inibiram as enzimas em estudo, porém, após a simulação do fluido gástrico o inibidor presente no chá verde perdeu a ação. O chá preto deixou de inibir a α-amilase e apresentou inibição inalterada para α e β-glicosidases. Tais resultados sugerem que o chá preto pode ser auxiliar em dietas de restrição de carboidratos.

Several plants have been considered therapeutic products, including green and black tea, popularly used to control hyperglycemia and obesity. This study aimed to evaluate the inhibitory potential of the enzymes α-amylase, α and β-glycosidases, as well as the content of phenolic compounds in green tea and black tea. The concentrations of phenolic compounds found were 80.8 ± 0.43 mg g-1 in black tea and 32.0 ± 0.12 mg g-1 in green tea. Under the tested conditions of use, green and black tea inhibited the enzyme under study. However, after simulation of the gastric fluid, the inhibitor present in green tea lost its action. Black tea no longer inhibited a-amylase and showed unaltered inhibition for α and β-glycosidases. These results suggest that black tea can be helpful in diets restricting carbohydrates.

No gender difference in effectiveness of smoking cessation treatment in a Brazilian real-life setting.

BACKGROUND: The clinical effectiveness of pharmacotherapy for smoking cessation in real-life settings has yet to be evaluated. OBJECTIVES: To assess the effectiveness of bupropion in general clinical practice for smoking cessation and to identify predictors of failure. METHODS: In an open, non-randomised study, smokers were recruited at the Smoking Cessation Clinics, Hospital Sao Lucas, Porto Alegre, Brazil. Subjects participated in a motivational group meeting, completed a standardised questionnaire and Fagerstrom test, and had their vital signs and exhaled CO registered. All participants received a prescription of bupropion and the same cognitive behaviour therapy. They attended eight weekly individual sessions, then monthly until the sixth month and a final session at month 12. The primary outcome measure was the rate of abstinence at 12 months. The predictor factors studied were sex, age, educational level, nicotine dependence, previous attempts and comorbidities. RESULTS: Among 253 smokers (62.5% females), abstinence rates at 6 months were 20.8% for males and 22.7% for females. The success rates dropped to 13.9% and 14.3% for males and females, respectively. CONCLUSIONS: Cognitive therapy plus bupropion for smoking cessation in real-life clinics in Brazil were similar to the efficacy found in clinical trials. No significant gender differences in success rates were found.

Use of proteolytic enzymes as an additional tool for trypanosomatid identification.

The expression of proteolytic activities in the Trypanosomatidae family was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from insects and plants. We have comparatively analysed the proteolytic profiles of 19 monoxenous trypanosomatids (Herpetomonas anglusteri, H. samuelpessoai, H. mariadeanei, H. roitmani, H. muscarum ingenoplastis, H. muscarum muscarum, H. megaseliae, H. dendoderi, Herpetomoas sp., Crithidia oncopelti, C. deanei, C. acanthocephali, C. harmosa, C. fasciculata, C. guilhermei, C. luciliae, Blastocrithidia culicis, Leptomonas samueli and Lept. seymouri) and 4 heteroxenous flagellates (Phytomonas serpens, P. mcgheei, Trypanosoma cruzi and Leishmania amazonensis) by in situ detection of enzyme activities on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE ) containing co-polymerized gelatine as substrate, in association with specific proteinase inhibitors. All 23 trypanosomatids expressed at least 1 acidic proteolytic enzyme. In addition, a characteristic and specific pattern of cell-associated metallo and/or cysteine proteinases was observed, except for the similar profiles detected in 2 Herpetomonas (H. anglusteri and H. samuelpessoai) and 3 Crithidia (C. fasciculata, C. guilhermei and C. luciliae) species. However, these flagellates released distinct secretory proteinase profiles into the extracellular medium. These findings strongly suggest that the association of cellular and secretory proteinase pattern could represent a useful marker to help trypanosomatid identification.

Cell-associated and extracellular proteinases in Blastocrithidia culicis: influence of growth conditions.

The proteinase profile of Blastocrithidia culicis was analyzed, as well as how different growth conditions influenced its expression by gelatin-SDS-PAGE and the use of specific proteinase inhibitors. Multiple cell-associated proteinases with molecular masses corresponding to 33, 55, 60 kDa (cysteine proteinases) and 77, 80, 90, and 108 kDa (metalloproteinases) were detected using these methods. All the metalloproteinases identified were partitioned into the detergent phase after Triton X-114 extract, suggesting that they are membrane-bound, while all cysteine proteinases were partitioned into the aqueous phase. The proteolytic zymograms were similar when three different media were used for variable times of incubation. However, few quantitative and qualitative changes were observed. The secreted proteinase profile showed an heterogeneous pattern of enzymatic activities whose expression was dependent on time of growth and medium composition. However, the extracellular proteinase activities were abolished by 1,10-phenanthroline, suggesting that all of them are zinc-metalloproteinases.

Developmentally regulated protein expression mediated by dimethylsulfoxide in Herpetomonas samuelpessoai.

We have analyzed the total cell extract, cell surface, and secretory protein profiles related to cellular differentiation triggered by dimethylsulfoxide in the insect trypanosomatid Herpetomonas samuelpessoai. The flagellates were cultivated in chemically defined conditions in the absence or in the presence of 4% DMSO, and the resolved protein bands were detected by SDS-PAGE gels and avidin-Western blotting. The cell-associated proteins showed a complex pattern of around 40 silver-staining bands ranging from 15 to 150 kDa. There were generally minor quantitative differences in the protein profile between the non-treated and the DMSO-treated cells. The cell-surface protein profile revealed by the incubation of live parasites with biotin showed a decrease in the expression of the 120 kDa biotinylated polypeptide observed in the DMSO-treated cells when compared with untreated ones. However, control samples of both systems showed an endogenous biotinylated polypeptide of 63 kDa which also presented gelatinolytic activity. The trypanosomatids released at least 10 polypeptides to the culture medium. A low molecular mass exopolypeptide (35 kDa) was found exclusively in untreated cells, whereas a high-molecular-mass exopolypetide (250 kDa) was preferentially found in DMSO-treated cells.

Detection of Trypanosoma-decay accelerating factor antibodies in mice and humans infected with Trypanosoma cruzi.

Highly purified Trypanosoma-decay accelerating factor (T-DAF), a 87-93-kD glycoprotein present on the surface of metacyclic and trypomastigote forms of Trypanosoma cruzi, was used as antigen to evaluate the presence of specific serum antibodies in experimentally infected mice and patients with Chagas' disease by enzyme-linked immunosorbent assay (ELISA). Mouse T-DAF antibodies were first recorded on day 7 postinfection, reached maximal concentration on day 30, and maintained at positive titers thereafter. High immunogenicity was clearly demonstrated by the detection of T-DAF antibodies in 96% of the sera collected from chagasic patients in either the acute or the chronic phase of disease. Control sera from normal individuals and from patients with leishmaniasis or other chronic infections did not give positive results. Serologic evaluation using T-DAF as antigen did not discriminate between patients with the cardiac and the digestive forms of the disease. The performance of the T-DAF ELISA was compared with that of conventional screening tests for Chagas' disease (indirect immunofluorescence and hemagglutination). The T-DAF ELISA test showed a sensitivity of 96%, a specificity of 100%, an efficiency of 99%, a positive predicted value of 100%, a negative predicted value of 98%, and a kappa index of 0.96, thus indicating that it can be successfully used for the serodiagnosis of T. cruzi infection in humans.

[2 infectious granulomatous diseases (leprosy and cutaneous and mucous leishmaniasis) by scintigraphic methods]. / Contribution au début de l'étude de deux maladies infectieuses granulomateuses (la lèpre et la leishmaniose cutanée et muqueuse) par des méthodes scintigraphiques.

Leprosy is a multiform chronic infectious granulomatous disease caused by Mycobacterium leprae, that affects over 12 million people in the world. Cutaneous and mucous leishmaniasis (CML) is also a chronic granulomatous infectious disease, caused by Leishmania brasiliensis and transmitted to man by the mosquitoes of the Phlebotominae family. It is a worldwide spread disease. We studied one case of Borderline-wirchowian leprosy and 2 cases of CML with Gallium-67 (GA-67) scintigraphy. Ga-67 is a radiopharmaceutical known for its property of concentrating in inflammatory sites. In the leprosy patient, Ga-67 accumulated in the skin in a moderate, homogeneous and disseminated way (outlined skin); in the area of the face, the uptake was important and homogeneous (image in beard). Several internal organs accumulated Ga-67. As for the 2 CML patients, Ga-67 accumulated focally, in different degrees, in the affected anatomical areas. The leprosy patient was not under treatment and the 2 CML were under treatment (20 and 40 days, respectively). In the 3 cases, all affected areas accumulated Ga-67. Intensity differences of uptake may be explained both by different degrees of inflammatory processes (between leprosy and CML) and by treatment lasting. It is possible that Ga-67 scintigraphy may be useful for the evaluation of these 2 diseases extent and also for the therapy follow-up.